Median fluorescence intensity of surface CXCR4 expression relative to isotype control ( n = 7 - 10 patients ) . Horizontal bars indicate mean of all individual patient values ; asterisks indicate statistical significance ( plasma cells vs B cells : P = 0 . 0082 ; plasma cells vs T cells : P = 0 . 0091 ; plasma cells vs CD34 + cells : P = 0 . 011 ; plasma cells vs monocytes : P = 0 . 0423 ; Student ' s t - test to compare mean plasmacell relative expression with mean relative expression of each further indicated cell subtype ) . MM patients judged positive for CXCR4 expression were selected for this analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Median fluorescence intensity of surface CXCR4 expression relative to isotype control (n = 7-10 patients). Horizontal bars indicate mean of all individual patient values; asterisks indicate statistical significance (plasma cells vs B cells: P = 0.0082; plasma cells vs T cells: P = 0.0091; plasma cells vs CD34+ cells: P = 0.011; plasma cells vs monocytes: P = 0.0423; Student's t-test to compare mean plasmacell relative expression with mean relative expression of each further indicated cell subtype). MM patients judged positive for CXCR4 expression were selected for this analysis.


Representative MM bone marrow staining of a patient positive for MM CXCR4 expression : hematoxylin and eosin ( H & amp ; amp ; E ) staining . Immunohistochemistry for CD138 , CXCR4 , light chain kappa , and light chain lambda .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative MM bone marrow staining of a patient positive for MM CXCR4 expression: hematoxylin and eosin (H&amp;E) staining. Immunohistochemistry for CD138, CXCR4, light chain kappa, and light chain lambda.


Real - time PCR analysis of cxcr4 transcript expression levels in HeLa ( negative control ) and in MM cell lines MM . 1S and OPM - 2 . Shown is the mean relative expression ± SEM ( n = 3 independent experiments ) . Values are normalized to the expression of ubiquitin ( Ub ) . The asterisk indicates statistically significant differences ( HeLa vs MM . 1S : P < 0 . 001 ; HeLa vs OPM - 2 : P < 0 . 0001 ; Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Real-time PCR analysis of cxcr4 transcript expression levels in HeLa (negative control) and in MM cell lines MM.1S and OPM-2. Shown is the mean relative expression ± SEM (n = 3 independent experiments). Values are normalized to the expression of ubiquitin (Ub). The asterisk indicates statistically significant differences (HeLa vs MM.1S: P < 0.001; HeLa vs OPM-2: P < 0.0001; Student's t-test).


CXCR4 protein expression assessed by immunoblotting ( one representative blot out of 3 is shown ) .

CXCR4 protein expression assessed by immunoblotting (one representative blot out of 3 is shown).


Binding of [ 68Ga ] Pentixafor to MM . 1S and OPM - 2 cells after the indicated incubation periods ( n = 3 per cell line and time point ) . Shown is the mean ± SEM . The difference between the OPM - 2 groups is statistically significant ; * P < 0 . 0017 ( one - way ANOVA ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Binding of [68Ga]Pentixafor to MM.1S and OPM-2 cells after the indicated incubation periods (n = 3 per cell line and time point). Shown is the mean ± SEM. The difference between the OPM-2 groups is statistically significant; *P < 0.0017 (one-way ANOVA).


Mean tumor - to - background ratio ( TBR ) for [ 18F ] FDG ( left ) and for [ 68Ga ] Pentixafor ( right ) in MM . 1S and OPM - 2 xenograft - bearing NOD SCID mice . Shown is the mean ± SEM , n = 8 tumors ( 4 mice ) ; * P = 0 . 0111 for [ 18F ] FDG and * P = 0 . 0113 for [ 68Ga ] Pentixafor ( Student ' s t - test ) . One - way ANOVA revealed significant differences between the groups ; P < 0 . 0001 ( not graphically shown ) .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean tumor-to-background ratio (TBR) for [18F]FDG (left) and for [68Ga]Pentixafor (right) in MM.1S and OPM-2 xenograft-bearing NOD SCID mice. Shown is the mean ± SEM, n = 8 tumors (4 mice); *P = 0.0111 for [18F]FDG and *P = 0.0113 for [68Ga]Pentixafor (Student's t-test). One-way ANOVA revealed significant differences between the groups; P < 0.0001 (not graphically shown).


Representative [ 68Ga ] Pentixafor PET images of three mice bearing MM . 1S ( right shoulder ) and OPM - 2 ( left shoulder ) tumors .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Representative [68Ga]Pentixafor PET images of three mice bearing MM.1S (right shoulder) and OPM-2 (left shoulder) tumors.


Flow cytometric quantification of CXCR4 cell surface expression on resected MM . 1S and OPM - 2 tumors . Data are the mean ± SEM , n = 4 . * P = 0 . 0286 ; Mann - Whitney U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Flow cytometric quantification of CXCR4 cell surface expression on resected MM.1S and OPM-2 tumors. Data are the mean ± SEM, n = 4. *P = 0.0286; Mann-Whitney U-test.


Correlation of [ 68Ga ] Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry . n = 8 tumors were analyzed .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

Correlation of [68Ga]Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry. n = 8 tumors were analyzed.


Mice ( n = 4 ) bearing OPM - 2 and MM . 1S xenografts were coinjected with AMD3100 ( right image , one representative mouse ) or not pretreated ( left image ) before undergoing [ 68Ga ] PentixaforPET . The white arrows point to the bladder . Quantification is shown in Supplementary Fig S3A . Source data are available online for this figure .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice (n = 4) bearing OPM-2 and MM.1S xenografts were coinjected with AMD3100 (right image, one representative mouse) or not pretreated (left image) before undergoing [68Ga]PentixaforPET. The white arrows point to the bladder. Quantification is shown in Supplementary Fig S3A.Source data are available online for this figure.


[ 68Ga ] PentixaforPET / CT and [ 18F ] FDGPET / CTA - D Maximum intensity projections ( MIP ) of [ 68Ga ] Pentixafor ( A ) and [ 18F ] FDGPET / CT ( B ) of a 68 - year - old male with histologically proven multiple myeloma indicating the better lesion - to - background contrast for [ 68Ga ] Pentixafor in the corresponding myeloma manifestations . Trans - axial views of the upper thorax ( C ) and the pelvis ( D ) underline the higher uptake values of the bone manifestations ( yellow arrows ) of [ 68Ga ] Pentixafor compared to [ 18F ] FDG .

O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O

[68Ga]PentixaforPET/CT and [18F]FDGPET/CTA-D Maximum intensity projections (MIP) of [68Ga]Pentixafor (A) and [18F]FDGPET/CT (B) of a 68-year-old male with histologically proven multiple myeloma indicating the better lesion-to-background contrast for [68Ga]Pentixafor in the corresponding myeloma manifestations. Trans-axial views of the upper thorax (C) and the pelvis (D) underline the higher uptake values of the bone manifestations (yellow arrows) of [68Ga]Pentixafor compared to [18F]FDG.


Visual comparison of [ 18F ] FDG - and [ 68Ga ] Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer ( total : n = 14 ) . Number of patients ( total n = 14 ) for whom imaging with [ 18F ] FDG PET ( FDG , n = 2 ) or [ 68Ga ] Pentixafor PET ( Pentixafor , n = 7 ) was superior , with comparable positivity ( comparable , n = 3 ) , and with dual imaging providing complementary visual information ( complementary , n = 2 ) .

O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Visual comparison of [18F]FDG- and [68Ga]Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer (total: n = 14).Number of patients (total n = 14) for whom imaging with [18F]FDG PET (FDG, n = 2) or [68Ga]Pentixafor PET (Pentixafor, n = 7) was superior, with comparable positivity (comparable, n = 3), and with dual imaging providing complementary visual information (complementary, n = 2).


[ 68Ga ] PentixaforPET / MRimagesA - C Coronal views of [ 68Ga ] Pentixafor ( A ) , T2 STIR weighted MRI ( B ) and CTbone window ( C ) of a maleuptake with histologically proven multiple myeloma . The increased [ 68Ga ] Pentixaforbone correlates with the hyperintense T2 STIR signal ; however , the myeloma manifestations are underestimated in the corresponding bone window CT .

O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

[68Ga]PentixaforPET/MRimagesA-C Coronal views of [68Ga]Pentixafor (A), T2 STIR weighted MRI (B) and CTbone window (C) of a maleuptake with histologically proven multiple myeloma. The increased [68Ga]Pentixaforbone correlates with the hyperintense T2 STIR signal; however, the myeloma manifestations are underestimated in the corresponding bone window CT.


( A ′ ) HeLa cells transfected with pEGFP - RAB11 were fixed in basal conditions and stained for mATG9 . Pearson ' s coefficient : 0 . 452 ± 0 . 034 ( SEM ) . ( A ″ ) HeLa cells were transfected with ATG9L1 - pEGFP , fixed in basal condition , and labeled for RAB11 . Pearson ' s coefficient : 0 . 413 ± 0 . 020 ( SEM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A′) HeLa cells transfected with pEGFP-RAB11 were fixed in basal conditions and stained for mATG9. Pearson's coefficient: 0.452 ± 0.034 (SEM).(A″) HeLa cells were transfected with ATG9L1-pEGFP, fixed in basal condition, and labeled for RAB11. Pearson's coefficient: 0.413 ± 0.020 (SEM).


( B ) HeLa cells were incubated for 1 hr at 37 ° C or 18 ° C to reach the correct temperature , loaded with transferrin Alexa - 555 for 1 hr , fixed , and labeled with anti - ATG9 and anti - TGN46 ( marker for Trans Golgi Network ) . Histogram shows Pearson ' s coefficient between ATG9 and transferrin ( Tf ) or TGN46 . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . Inserts show merge at higher magnification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) HeLa cells were incubated for 1 hr at 37°C or 18°C to reach the correct temperature, loaded with transferrin Alexa-555 for 1 hr, fixed, and labeled with anti-ATG9 and anti-TGN46 (marker for Trans Golgi Network). Histogram shows Pearson's coefficient between ATG9 and transferrin (Tf) or TGN46. Error bar, SEM. ∗∗∗p < 0.001. Inserts show merge at higher magnification.


( C ) HeLa cells were incubated in serum - free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa - 555 . Cells were then fixed and labeled for mATG9 and TGN46 . Pearson ' s coefficient between mATG9 and transferrin or TGN46 was quantified . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . Inserts show the merge at higher magnification .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) HeLa cells were incubated in serum-free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa-555. Cells were then fixed and labeled for mATG9 and TGN46. Pearson's coefficient between mATG9 and transferrin or TGN46 was quantified. Error bar, SEM. ∗∗∗p < 0.001. Inserts show the merge at higher magnification.


( D ) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy . Pictures are at the same magnification and brightness . Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy. Pictures are at the same magnification and brightness. Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane.


( E ) HeLa cells were incubated for 1 hr at 4 ° C with ice - cold NHS - LC - Biotin ( Pierce ) solution , rinsed , and lysed in RIPA buffer . Biotinylated proteins were precipitated using streptavidin - agarose beads ( Pierce ) and blotted for mATG9 to assess protein localization at the plasma membrane .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) HeLa cells were incubated for 1 hr at 4°C with ice-cold NHS-LC-Biotin (Pierce) solution, rinsed, and lysed in RIPA buffer. Biotinylated proteins were precipitated using streptavidin-agarose beads (Pierce) and blotted for mATG9 to assess protein localization at the plasma membrane.


( F ) HeLa cells treated as in ( C ) were processed for immunogold labeling on cryosections and stained with anti - mATG9 antibody . Arrows show clathrin - coated structures . Scale bar in a and c , 150 nm ; scale bar in b , 100 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) HeLa cells treated as in (C) were processed for immunogold labeling on cryosections and stained with anti-mATG9 antibody. Arrows show clathrin-coated structures. Scale bar in a and c, 150 nm; scale bar in b, 100 nm.


( G ) HeLa cells transfected with GFP - ATG16L1 were fixed , labeled for mATG9 , and then analyzed by TIRF microscopy . Control cells were cotransfected with GFP - ATG16L1 and Straw - ATG16L1 . Pictures are at the same magnification and brightness . See Figure S1J for quantification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) HeLa cells transfected with GFP-ATG16L1 were fixed, labeled for mATG9, and then analyzed by TIRF microscopy. Control cells were cotransfected with GFP-ATG16L1 and Straw-ATG16L1. Pictures are at the same magnification and brightness. See Figure S1J for quantification.


( H ) HeLa cells treated with Dynasore were processed for immunogold labeling on cryosections and double labeled with ATG16L1 ( 10 nm ) and mATG9 ( 15 nm ) . Scale bar , 100 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) HeLa cells treated with Dynasore were processed for immunogold labeling on cryosections and double labeled with ATG16L1 (10 nm) and mATG9 (15 nm). Scale bar, 100 nm.


( I ) HeLa cells treated as in ( H ) were processed for triple labeling on cryosections ( clathrin , 5 nm ; ATG16L1 , 10 nm ; and mATG9 , 15 nm ) . Scale bar , 150 nm . ( J ) Table shows quantification of numbers of clathrin - coated structures carrying mATG9 and ATG16L1 alone or together or neither protein . A minimum of 50 clathrin - coated structures were examined from three different experiments . We constructed a 2 × 2 table with 0 or 1 - 3 gold particles for mATG9 or ATG16L1 for a X2 test to avoid statistical cells with 0 counts . X2 = 10 . 4 , df = 1 , and p = 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) HeLa cells treated as in (H) were processed for triple labeling on cryosections (clathrin, 5 nm; ATG16L1, 10 nm; and mATG9, 15 nm). Scale bar, 150 nm.(J) Table shows quantification of numbers of clathrin-coated structures carrying mATG9 and ATG16L1 alone or together or neither protein. A minimum of 50 clathrin-coated structures were examined from three different experiments. We constructed a 2 × 2 table with 0 or 1-3 gold particles for mATG9 or ATG16L1 for a X2 test to avoid statistical cells with 0 counts. X2 = 10.4, df = 1, and p = 0.001.


( A ) HeLa cells transfected with GFP - ATG16L1 for 24 hr were incubated for 15 min with 2 . 5 μg / ml with HRP - cholera toxin subunit B at 4 ° C and then for 10 min at 37 ° C . Cells were then fixed and treated for immunogold labeling on cryosections . ATG16L1 was detected with anti - ATG16L1 antibody ( 15 nm ) , and cholera toxin was detected using anti - HRP antibody . Scale bar , 150 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HeLa cells transfected with GFP-ATG16L1 for 24 hr were incubated for 15 min with 2.5 μg/ml with HRP-cholera toxin subunit B at 4°C and then for 10 min at 37°C. Cells were then fixed and treated for immunogold labeling on cryosections. ATG16L1 was detected with anti-ATG16L1 antibody (15 nm), and cholera toxin was detected using anti-HRP antibody. Scale bar, 150 nm.


( B ) HeLa cells were transiently transfected with GFP - ATG16L1 for 20 hr . Cells in basal conditions were fixed and labeled with anti - RAB11 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) HeLa cells were transiently transfected with GFP-ATG16L1 for 20 hr. Cells in basal conditions were fixed and labeled with anti-RAB11 antibody.


( C ) HeLa cells transfected with mStrawberry - ATG16L1 ( 20 hr ) and starved ( 4 hr ) were processed for immunogold labeling on cryosections . Double labeling was performed with rabbit anti - ATG16L1 ( 10 nm gold ) and rabbit anti - RAB11 ( 15 nm gold ) . Scale bar , 150 nm . Schematic diagram shows membrane outlines from EM . Green lines indicate structures positive for both ATG16L1 and RAB11 , whereas red lines are only positive for RAB11 . Histogram shows the percentage by EM of ATG16L1 tubulovesicular structures that carry RAB11 . 281 tubulovesiclular structures from different experiments were analyzed . Error bars , SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) HeLa cells transfected with mStrawberry-ATG16L1 (20 hr) and starved (4 hr) were processed for immunogold labeling on cryosections. Double labeling was performed with rabbit anti-ATG16L1 (10 nm gold) and rabbit anti-RAB11 (15 nm gold). Scale bar, 150 nm. Schematic diagram shows membrane outlines from EM. Green lines indicate structures positive for both ATG16L1 and RAB11, whereas red lines are only positive for RAB11. Histogram shows the percentage by EM of ATG16L1 tubulovesicular structures that carry RAB11. 281 tubulovesiclular structures from different experiments were analyzed. Error bars, SD.


( D ) HeLa cells were transfected with EGFP - ATG16L1 and mCherry - RAB11 , and 5 min movies were recorded . Representative images from movies are shown .

(D) HeLa cells were transfected with EGFP-ATG16L1 and mCherry-RAB11, and 5 min movies were recorded. Representative images from movies are shown.


( E ) HeLa cells were loaded with mouse anti - transferrin receptor antibody ( 5E9C11 ) for 30 min and fixed for immunogold labeling for cryosections . The sample was labeled with rabbit anti - mouse antibody to recognize transferrin receptor ( 5 nm ) , anti - ATG16L1 ( 10 nm ) , and anti - mATG9 ( 15 nm ) . Scale bar , 150 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) HeLa cells were loaded with mouse anti-transferrin receptor antibody (5E9C11) for 30 min and fixed for immunogold labeling for cryosections. The sample was labeled with rabbit anti-mouse antibody to recognize transferrin receptor (5 nm), anti-ATG16L1 (10 nm), and anti-mATG9 (15 nm). Scale bar, 150 nm.


( F ) HeLa cells were transfected with pmStrawberry - ATG16L1 and ATG9L1 - pEGFP , and 5 min movies were recorded . Representative images from the movies are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) HeLa cells were transfected with pmStrawberry-ATG16L1 and ATG9L1-pEGFP, and 5 min movies were recorded. Representative images from the movies are shown.


( G ) HeLa cells were transfected with pmStrawberry - ATG16L1 and pEGFP - LC3 , starved for 2 hr , fixed , and labeled for mATG9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) HeLa cells were transfected with pmStrawberry-ATG16L1 and pEGFP-LC3, starved for 2 hr, fixed, and labeled for mATG9.


( A and B ) ( A ) HeLa cells were incubated for 4 hr at 37 ° C or 18 ° C and processed for LC3II western blot . Some cells were incubated overnight with 20 mM NH4Cl in full medium ( to block lysosomal degradation ) at 37 ° C or 18 ° C ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(A and B) (A) HeLa cells were incubated for 4 hr at 37°C or 18°C and processed for LC3II western blot. Some cells were incubated overnight with 20 mM NH4Cl in full medium (to block lysosomal degradation) at 37°C or 18°C (B).


( C ) HeLa cells were transfected with pEGFP - LC3 for 20 hr and incubated for 4 hr at 37 ° C or 18 ° C .

(C) HeLa cells were transfected with pEGFP-LC3 for 20 hr and incubated for 4 hr at 37°C or 18°C.


( D and E ) ( D ) HeLa cells were transfected with pEGFP - ATG16L1 for 20 hr and incubated for 4 hr at 37 ° C or 18 ° C . The size and the number ( E ) of vesicles were scored ( a minimum of 20 cells were examined for each condition ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D and E) (D) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr and incubated for 4 hr at 37°C or 18°C. The size and the number (E) of vesicles were scored (a minimum of 20 cells were examined for each condition). Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01.


( F and G ) ( F ) HeLa cells were transfected with pEGFP - ATG16L1 for 20 hr , incubated for 4 hr at 37 ° C or 18 ° C , and labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes . Histogram in ( G ) shows the amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment ( Manders ' coefficient ) . Error bar , SEM . NS , not significant ; ∗ ∗ p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F and G) (F) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr, incubated for 4 hr at 37°C or 18°C, and labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes. Histogram in (G) shows the amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment (Manders' coefficient). Error bar, SEM. NS, not significant; ∗∗p < 0.01.


( H ) HeLa cells were transfected with pmStrawberry - ATG16L1 for 20 hr , fixed , and labeled for mATG9 . The histograms show quantification of colocalization and the size of the vesicles labeled with ATG16L1 and mATG9 at 37 ° C or 18 ° C . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ p < 0 . 01 .

(H) HeLa cells were transfected with pmStrawberry-ATG16L1 for 20 hr, fixed, and labeled for mATG9. The histograms show quantification of colocalization and the size of the vesicles labeled with ATG16L1 and mATG9 at 37°C or 18°C. Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01.


( I ) HeLa cells were transfected with pEGFP - RAB11 or pEGFP - Myosin Vb tail ( MVb ) and incubated during the last 16 hr with Bafilomycin A1 ( Baf ) or DMSO and processed for LC3 - II western blot . Control cells were transfected with pEGFP empty vector .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(I) HeLa cells were transfected with pEGFP-RAB11 or pEGFP-Myosin Vb tail (MVb) and incubated during the last 16 hr with Bafilomycin A1 (Baf) or DMSO and processed for LC3-II western blot. Control cells were transfected with pEGFP empty vector.


( J ) HeLa cells were transfected with ATG16L1 - mStrawberry and RAB11 - EGFP or MVb tail - EGFP . The size of ATG16L1 vesicles was scored in RAB11 and MVb tail overexpression conditions . The inserts show just the ATG16L1 vesicles as quantified in histogram . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) HeLa cells were transfected with ATG16L1-mStrawberry and RAB11-EGFP or MVb tail-EGFP. The size of ATG16L1 vesicles was scored in RAB11 and MVb tail overexpression conditions. The inserts show just the ATG16L1 vesicles as quantified in histogram. Error bar, SEM. ∗∗∗p<0.001.


( K ) HeLa cells were transfected as in ( C ) and loaded with transferrin Alexa - 647 for 30 min . Histogram and the inserts show the correlation between ATG16L1 and the loaded transferrin ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) HeLa cells were transfected as in (C) and loaded with transferrin Alexa-647 for 30 min. Histogram and the inserts show the correlation between ATG16L1 and the loaded transferrin (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( L ) HeLa cells were transfected as in ( C ) and labeled for mATG9 . Histogram and the inserts show the correlation between ATG16L1 and mATG9 in RAB11 and MVb tail overexpression conditions ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) HeLa cells were transfected as in (C) and labeled for mATG9. Histogram and the inserts show the correlation between ATG16L1 and mATG9 in RAB11 and MVb tail overexpression conditions (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( M ) HeLa cells were transfected with mStrawberry - ATG16L1 for 20 hr , starved in HBSS for 4 hr , or maintained in full medium , loaded with transferrin Alexa - 488 ( Tf ) . Pictures and histogram show the correlation between ATG16L1 and transferrin in basal and starving condition ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium, loaded with transferrin Alexa-488 (Tf). Pictures and histogram show the correlation between ATG16L1 and transferrin in basal and starving condition (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( N ) HeLa cells were transfected with mStrawberry - ATG16L1 for 20 hr , starved in HBSS for 4 hr , or maintained in full medium and then fixed and labeled with anti - mATG9 antibody . Pictures and histogram show the correlation between ATG16L1 and mATG9 in basal and starved cells ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 .

(N) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium and then fixed and labeled with anti-mATG9 antibody. Pictures and histogram show the correlation between ATG16L1 and mATG9 in basal and starved cells (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( A ) HeLa cells were transfected with mStrawberry - ATG16 for 20 hr and treated for 10 min with N - ethylmaleimide ( NEM ; 100 μM ) in full medium , fixed , and stained for mATG9 . Histogram shows correlation between mATG9 and ATG16L1 ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HeLa cells were transfected with mStrawberry-ATG16 for 20 hr and treated for 10 min with N-ethylmaleimide (NEM; 100 μM) in full medium, fixed, and stained for mATG9. Histogram shows correlation between mATG9 and ATG16L1 (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( C and D ) ( C ) Representative fields of in vitro fusion assay of ATG16L1 and mATG9 vesicles . The quantification in ( D ) was performed on ten fields ( similar to that shown in the figure ) per experiment . The quantification is expressed as Pearson ' s coefficient . Error bar , SEM . ∗ ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C and D) (C) Representative fields of in vitro fusion assay of ATG16L1 and mATG9 vesicles. The quantification in (D) was performed on ten fields (similar to that shown in the figure) per experiment. The quantification is expressed as Pearson's coefficient. Error bar, SEM. ∗∗p < 0.001.


( A ) HeLa cells were transfected with siRNAs for different R - SNAREs , and we assessed the levels of the autophagy substrate p62 versus actin .

(A) HeLa cells were transfected with siRNAs for different R-SNAREs, and we assessed the levels of the autophagy substrate p62 versus actin.


( B ) HeLa cells transfected with control or VAMP3 siRNA ( oligo A ) for 4 days were treated during the last 16 hr with Bafilomycin A1 ( Baf A1 ) or DMSO ( basal ) and processed for LC3 - II western blots .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

(B) HeLa cells transfected with control or VAMP3 siRNA (oligo A) for 4 days were treated during the last 16 hr with Bafilomycin A1 (Baf A1) or DMSO (basal) and processed for LC3-II western blots.


( C ) Endogenous LC3 immunocytochemistry in HeLa cells transfected with control or VAMP3 siRNA for 4 days . The histogram shows the number of vesicles ( LC3 positive ) in a minimum of 30 cells . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Endogenous LC3 immunocytochemistry in HeLa cells transfected with control or VAMP3 siRNA for 4 days. The histogram shows the number of vesicles (LC3 positive) in a minimum of 30 cells. Error bar, SEM. ∗∗∗p < 0.001.


( D ) HeLa cells were transfected with VAMP3 - HA for 20 hr and stained for EEA1 or RAB11 . Histogram shows colocalization between VAMP3 - HA and EEA1 or VAMP3 - HA and RAB11 ( Manders ' coefficient ) . Error bar , SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) HeLa cells were transfected with VAMP3-HA for 20 hr and stained for EEA1 or RAB11. Histogram shows colocalization between VAMP3-HA and EEA1 or VAMP3-HA and RAB11 (Manders' coefficient). Error bar, SEM.


( E ) HeLa cells were transfected for 20 hr with VAMP3 - HA , incubated for 1 hr at 37 ° C or 18 ° C , fixed , and labeled for HA and mATG9 . Histogram shows the percentage of colocalization between VAMP3 with mATG9 ( Manders ' coefficient ) . Error bar , SEM . NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) HeLa cells were transfected for 20 hr with VAMP3-HA, incubated for 1 hr at 37°C or 18°C, fixed, and labeled for HA and mATG9. Histogram shows the percentage of colocalization between VAMP3 with mATG9 (Manders' coefficient). Error bar, SEM. NS, not significant.


( A - C ) HeLa cells transfected with control , VAMP3 siRNA for 4 days were transfected during the last 20 hr with ATG9L1 - pEGFP and labeled with anti - EEA1 ( for early endosomes ) or anti - RAB11 ( for recycling endosomes ) . The histogram in ( C ) shows the quantification of the colocalization of mATG9 in EEA1 or RAB11 ( Manders ' coefficient ) . Error bars , SEM . ∗ ∗ ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with ATG9L1-pEGFP and labeled with anti-EEA1 (for early endosomes) or anti-RAB11 (for recycling endosomes). The histogram in (C) shows the quantification of the colocalization of mATG9 in EEA1 or RAB11 (Manders' coefficient). Error bars, SEM. ∗∗∗p < 0.001.


( D - F ) HeLa cells were treated as in ( A ) - ( C ) and transfected during the last 20 hr with pEGFP - ATG16L1 and labeled with anti - EEA1 ( early endosomes ) or anti - RAB11 ( recycling endosomes ) . Histogram in ( D ) shows quantification of the colocalization of ATG16L1 in EEA1 or RAB11 ( Manders ' coefficient ) . Error bars , SEM . ∗ ∗ ∗ p < 0 . 001 ; NS , not significant .

(D-F) HeLa cells were treated as in (A)-(C) and transfected during the last 20 hr with pEGFP-ATG16L1 and labeled with anti-EEA1 (early endosomes) or anti-RAB11 (recycling endosomes). Histogram in (D) shows quantification of the colocalization of ATG16L1 in EEA1 or RAB11 (Manders' coefficient). Error bars, SEM. ∗∗∗p < 0.001; NS, not significant.


( A ) HeLa cells transfected with control , VAMP3 siRNA for 4 days were transfected during the last 20 hr with mStrawberry - ATG16L1 and labeled for mATG9 . The correlation between mATG9 and ATG16L1 was quantified ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with mStrawberry-ATG16L1 and labeled for mATG9. The correlation between mATG9 and ATG16L1 was quantified (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( B ) HeLa cells were transfected with control or VAMP3 siRNA and then with mStrawberry - ATG16 , and 5 min movies were recorded . Homotypic fusion events between mStrawberry - ATG16 vesicles were assessed in control and VAMP3 knockdown cells . Error bar , SEM . NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) HeLa cells were transfected with control or VAMP3 siRNA and then with mStrawberry-ATG16, and 5 min movies were recorded. Homotypic fusion events between mStrawberry-ATG16 vesicles were assessed in control and VAMP3 knockdown cells. Error bar, SEM. NS, not significant.


( C ) In vitro fusion assay of ATG16L1 and mATG9 vesicles . HeLa cells were transfected with control or VAMP3 siRNA for 4 days separately and , on the fourth day , either transfected with mStrawberry - ATG16L1 or ATG9L1 - pEGFP . PNS of cells expressing the two constructs were mixed for 1 hr at 37C ° and observed by confocal microscopy . Histogram shows the correlation of vesicles carrying ATG16L1 that fuse with vesicles carrying mATG9 ( Pearson ' s coefficient ) . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . The total number of structures per field ( ATG16L1 and ATG9L1 ) is not significantly different in the control and KD samples ( control , 37 ± 3 ; VAMP3 KD , 45 ± 3 ; ± SEM ; p = 0 . 106 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) In vitro fusion assay of ATG16L1 and mATG9 vesicles. HeLa cells were transfected with control or VAMP3 siRNA for 4 days separately and, on the fourth day, either transfected with mStrawberry-ATG16L1 or ATG9L1-pEGFP. PNS of cells expressing the two constructs were mixed for 1 hr at 37C° and observed by confocal microscopy. Histogram shows the correlation of vesicles carrying ATG16L1 that fuse with vesicles carrying mATG9 (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001. The total number of structures per field (ATG16L1 and ATG9L1) is not significantly different in the control and KD samples (control, 37 ± 3; VAMP3 KD, 45 ± 3; ± SEM; p = 0.106).


( A ) THP - 1 cells were subjected to knockdowns as indicated and treated with 100 ng / mL LPS overnight , followed by 0 . 25 mM LLOMe for 3 h , and the levels of IL - 1β were determined in supernatants .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(A) THP-1 cells were subjected to knockdowns as indicated and treated with 100 ng/mL LPS overnight, followed by 0.25 mM LLOMe for 3 h, and the levels of IL-1β were determined in supernatants.


( B ) THP - 1 cells were subjected to GABARAP knockdown and processed as in A .

(B) THP-1 cells were subjected to GABARAP knockdown and processed as in A.


( C ) IL - 1β levels in supernatants of THP - 1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe.


( D ) Confocal microscopy of THP - 1 cells treated sequentially with LPS and LLOMe , and stained for LC3B and TRIM16 . Line tracings correspond to arrows . Arrowheads indicate colocalization . Scale bars , 5 μm .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(D) Confocal microscopy of THP-1 cells treated sequentially with LPS and LLOMe, and stained for LC3B and TRIM16. Line tracings correspond to arrows. Arrowheads indicate colocalization. Scale bars, 5 μm.


( E ) IL - 1β levels in supernatants of THP - 1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

(E) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe.


( F ) Levels of IL - 1β were determine in supernatants from THP - 1 cells subjected to knockdown as indicated , treated with 100 ng / mL LPS for overnight and then with 0 . 25mM of Silica , Alum , or monosodium urate ( MSU ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O

(F) Levels of IL-1β were determine in supernatants from THP-1 cells subjected to knockdown as indicated, treated with 100 ng/mL LPS for overnight and then with 0.25mM of Silica, Alum, or monosodium urate (MSU).


( G ) IL - 1β in supernatants of primary human macrophages ( MDM ) subjected to knockdowns and sequentially treated with LPS and LLOMe . Data , means ± SEM ; n ≥ 5 ( except for B where a representative data set from 3 repeats ) . * P < 0 . 05 ( t test or ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. Data, means ± SEM; n≥5 (except for B where a representative data set from 3 repeats). *P < 0.05 (t test or ANOVA).


( A and B ) Levels of IL - 1β in supernatants from THP - 1 cells that were subjected to knockdown as indicated and were treated with LPS , and then ( A ) treated with LLOMe or ( B ) starved in EBSS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(A and B) Levels of IL-1β in supernatants from THP-1 cells that were subjected to knockdown as indicated and were treated with LPS, and then (A) treated with LLOMe or (B) starved in EBSS.


( C ) IL - 1β in supernatants of primary human macrophages ( MDM ) subjected to knockdowns and sequentially treated with LPS and LLOMe .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

(C) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe.


( D ) Co - immunoprecipitation ( co - IP ) of flag - galectin - 8 ( Gal - 8 ) and GFP - TRIM16 ( T16 ) in HEK293T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Co-immunoprecipitation (co-IP) of flag-galectin-8 (Gal-8) and GFP-TRIM16 (T16) in HEK293T cells.


( E ) Co - IP analysis of endogenous galectin - 8 and TRIM16 in protein complexes from LLOMe - treated THP - 1 cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Co-IP analysis of endogenous galectin-8 and TRIM16 in protein complexes from LLOMe-treated THP-1 cells.


( F ) Confocal microscopy of THP - 1 cells treated with LPS and LLOMe , and stained for TRIM16 and galectin - 8 . Line tracings correspond to arrowed dashed lines . Pearson correlation coefficient was calculated using SlideBook as described previously . Scale bars , 5 μm .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Confocal microscopy of THP-1 cells treated with LPS and LLOMe, and stained for TRIM16 and galectin-8. Line tracings correspond to arrowed dashed lines. Pearson correlation coefficient was calculated using SlideBook as described previously. Scale bars, 5 μm.


( G ) In vitro translated and radiolabeled [ 35S ] myc - HA - TRIM16 was incubated with GST - galectin - 8 in the presence ( + ) or absence ( - ) of flag - ULK1 and cold ATP , GST pulldowns were performed , and [ 35S ] radiolabeled Myc - HA - TRIM16 in pulled - down material detected by PAGE and autoradiography . Amounts of GST fusion proteins are shown in coommassie brilliant blue ( CBB ) - stained gels . Data , means  SEM ; n ≥ 5 , except for immunoblot quantifications where n ≥ 3 . * P < 0 . 05 , † P ≥ 0 . 05 ( t test or ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) In vitro translated and radiolabeled [35S] myc-HA-TRIM16 was incubated with GST-galectin-8 in the presence (+) or absence (-) of flag-ULK1 and cold ATP, GST pulldowns were performed, and [35S] radiolabeled Myc-HA-TRIM16 in pulled-down material detected by PAGE and autoradiography. Amounts of GST fusion proteins are shown in coommassie brilliant blue (CBB)-stained gels. Data, means  SEM; n≥5, except for immunoblot quantifications where n≥3. *P < 0.05, †P ≥ 0.05 (t test or ANOVA).


( A and B ) Immunoblot analyses of indicated membrane fractions prepared by differential centrifugation . HeLa cells reconstituted with flag - pro - IL - 1β and myc - pro - Caspase - 1 were treated with 1mM LLOMe for 1h , and lysates were subjected to differential centrifugations at 3 , 000xg ( 3k ) , 25 , 000xg ( 25k ) and 100 , 000xg ( 100k ) as depicted in the cartoon . Arrows , two ways the membranes were analyzed - pellets from velocity sedimentation were analyzed in panels B - D , or by further sucrose and OptiPrep density separation of 25k pellets in panels F and G .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A and B) Immunoblot analyses of indicated membrane fractions prepared by differential centrifugation. HeLa cells reconstituted with flag-pro-IL-1β and myc-pro-Caspase-1 were treated with 1mM LLOMe for 1h, and lysates were subjected to differential centrifugations at 3,000xg (3k), 25,000xg (25k) and 100,000xg (100k) as depicted in the cartoon. Arrows, two ways the membranes were analyzed - pellets from velocity sedimentation were analyzed in panels B-D, or by further sucrose and OptiPrep density separation of 25k pellets in panels F and G.


( C ) IL - 1β levels in 25k membrane fractions ( pellets ) from HeLa cells and their CRISPR knockout derivatives ( TRIM16KO ) that were reconstituted for IL - 1β secretion and treated with 1mM LLOMe for 1 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) IL-1β levels in 25k membrane fractions (pellets) from HeLa cells and their CRISPR knockout derivatives (TRIM16KO) that were reconstituted for IL-1β secretion and treated with 1mM LLOMe for 1 h.


( D ) Quantification of analyses in ( B ) . RI , relative intensity .

(D) Quantification of analyses in (B). RI, relative intensity.


( E ) Whole cell lysates ( immunoblots ) analyzed in F and G .

(E) Whole cell lysates (immunoblots) analyzed in F and G.


( F , G ) Sucrose density gradient analysis of 25k membranes . Note that mature IL - 1β ( mIL - 1β ) is recruited to and co - fractionates with TRIM16 + , Sec22b + , and LC3 - II + membranes in TRIM16wt HeLa cells reconstituted with pro - IL - 1β and pro - caspase 1 , but not in TRIM16KO mutant cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F,G) Sucrose density gradient analysis of 25k membranes. Note that mature IL-1β (mIL-1β) is recruited to and co-fractionates with TRIM16+, Sec22b+, and LC3-II+ membranes in TRIM16wt HeLa cells reconstituted with pro-IL-1β and pro-caspase 1, but not in TRIM16KO mutant cells.


( H ) Confocal images of LC3 in HeLa cells expressing GFP - Sec22b treated with LLOMe or untreated ( control ) . Arrowheads , colocalization . Scale bars , 5 μm . ( I ) GFP - Sec22b and LC3 overlapping area obtained by high content microscopy .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Confocal images of LC3 in HeLa cells expressing GFP-Sec22b treated with LLOMe or untreated (control). Arrowheads, colocalization. Scale bars, 5 μm. (I) GFP-Sec22b and LC3 overlapping area obtained by high content microscopy.


( J ) Co - IP analysis between flag - TRIM16 with GFP - Sec22b or GFP - syntaxin - 17 co - expressed in HEK293T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Co-IP analysis between flag-TRIM16 with GFP-Sec22b or GFP-syntaxin-17 co-expressed in HEK293T cells.


( K ) Co - IP analysis of endogenous proteins , TRIM16 and Sec22b , in lysates from THP - 1 cells treated with LLOMe .

(K) Co-IP analysis of endogenous proteins, TRIM16 and Sec22b, in lysates from THP-1 cells treated with LLOMe.


( L ) Co - IP analysis of interactions between deletion variants of flag - TRIM16 with GFP - Sec22b in HEK293T cells .

O O O O O O O O O O O O O O O O O O O O O O O O

(L) Co-IP analysis of interactions between deletion variants of flag-TRIM16 with GFP-Sec22b in HEK293T cells.


( M and N ) IL - 1β secretion complementation experiments of TRIM16KO cells with TRIM16 Sec22b - interacting and its Sec22b - nonbinding mutant shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M and N) IL-1β secretion complementation experiments of TRIM16KO cells with TRIM16 Sec22b-interacting and its Sec22b-nonbinding mutant shown.


( O ) Two color super - resolution image showing co - clustering of flag - TRIM16 ( red ) and GFP - Sec22b ( green ) ; Scale bar , 200 nm . ( P ) Cross - correlation analysis of flag - TRIM16 and GFP - Sec22b super - resolution data showing a characteristic separation of ~ 70 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(O) Two color super-resolution image showing co-clustering of flag-TRIM16 (red) and GFP-Sec22b (green); Scale bar, 200 nm. (P) Cross-correlation analysis of flag-TRIM16 and GFP-Sec22b super-resolution data showing a characteristic separation of ~ 70 nm.


( A and B ) IL - 1β levels in supernatants of THP - 1 cells subjected to knockdowns , treated with LPS , and then ( A ) with LLOMe or ( B ) starved in EBSS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(A and B) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then (A) with LLOMe or (B) starved in EBSS.


( C ) IL - 1β levels in supernatants of primary human macrophages ( MDM ) subjected to knockdowns and sequentially treated with LPS and LLOMe .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

(C) IL-1β levels in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe.


( D ) Immunoblot analyses of p62 levels in THP - 1 cells subjected to knockdowns as indicated and starved for 3 h .

(D) Immunoblot analyses of p62 levels in THP-1 cells subjected to knockdowns as indicated and starved for 3 h.


( E ) mRFP - GFP overlapping areas of tandem HeLa cells reconstituted with flag - pro - IL - 1β and myc - Caspase 1 and starved for 3 h . Mask overlay , iDev software - defined objects ( primary objects , cell outlines ; internal secondary objects , GFP ( top ) , mRFP ( middle ) , or their overlapping area ( bottom ) ) . Bars , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) mRFP-GFP overlapping areas of tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 and starved for 3 h. Mask overlay, iDev software-defined objects (primary objects, cell outlines; internal secondary objects, GFP (top), mRFP (middle), or their overlapping area (bottom)). Bars, 10 μm.


( F ) IL - 1β levels in supernatants of starved tandem HeLa cells reconstituted with flag - pro - IL - 1β and myc - Caspase 1 as in E .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) IL-1β levels in supernatants of starved tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 as in E.


( A - C ) IL - 1β levels in supernatants of THP - 1 cells subjected to knockdowns , treated with LPS , and then with LLOMe ( A and C ) or starved in EBSS ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C) or starved in EBSS (B).


( A - C ) IL - 1β levels in supernatants of THP - 1 cells subjected to knockdowns , treated with LPS , and then with LLOMe ( A and C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C).


( C ) Ferritin levels in supernatants of THP - 1 cells subjected to knockdowns as indicated and sequentially treated with LPS and LLOMe .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

(C) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and sequentially treated with LPS and LLOMe.


( D ) Ferritin levels in supernatants of THP - 1 cells subjected to knockdowns as indicated and infected with M . tuberculosis as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and infected with M. tuberculosis as indicated.


( E ) Levels of ferritin were determine in supernatants from THP - 1 cells that were subjected to knockdown as indicated and were sequentially treated with LPS and LLOMe .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

(E) Levels of ferritin were determine in supernatants from THP-1 cells that were subjected to knockdown as indicated and were sequentially treated with LPS and LLOMe.


( F ) Confocal microscopy of HeLa cells expressing GFP - Sec22b were treated with LLOMe , and stained for FTH1 . Line tracings correspond to arrows . Scale bars , 5 μm ;

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

(F) Confocal microscopy of HeLa cells expressing GFP-Sec22b were treated with LLOMe, and stained for FTH1. Line tracings correspond to arrows. Scale bars, 5 μm;


( F ) Malate dehydrogenase ( MDH ; 0 . 2 µM ) was incubated at 40 ° C in the absence ( black ) or presence of 0 . 5 µM WT HSP27 ( red ) or 0 . 5 µM P182L HSP27 ( blue ) . ( G ) Insulin ( 40 µM ) was incubated at 40 ° C in the absence ( black ) or presence of 20 µM WT HSP27 ( red ) or 20 µM P182L HSP27 ( blue ) . The y - axes depict the absorbance at 340 nm due to the formation of large aggregates . The solid lines represent the average of three replicates with the filled area reflecting ± one standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Malate dehydrogenase (MDH; 0.2 µM) was incubated at 40 °C in the absence (black) or presence of 0.5 µM WT HSP27 (red) or 0.5 µM P182L HSP27 (blue). (G) Insulin (40 µM) was incubated at 40 °C in the absence (black) or presence of 20 µM WT HSP27 (red) or 20 µM P182L HSP27 (blue). The y-axes depict the absorbance at 340 nm due to the formation of large aggregates. The solid lines represent the average of three replicates with the filled area reflecting ± one standard deviation.


( A ) SEC - MALS data from WT HSP27 and the P182L variant , with cHSP27 included for comparison . All samples were injected at 40 µM concentration ( monomer ) . The respective average molecular masses are 14 MDa , 470 kDa , and 18 kDa for P182L , WT HSP27 , and cHSP27 . The normalized UV signals are plotted on the left y - axis as solid lines and the molecular masses are plotted on the right y - axis as circles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) SEC-MALS data from WT HSP27 and the P182L variant, with cHSP27 included for comparison. All samples were injected at 40 µM concentration (monomer). The respective average molecular masses are 14 MDa, 470 kDa, and 18 kDa for P182L, WT HSP27, and cHSP27. The normalized UV signals are plotted on the left y-axis as solid lines and the molecular masses are plotted on the right y-axis as circles.


( B ) Fractionation of HeLa cells overexpressing either HSP27 or the P182L mutant by a 10 % to 80 % sucrose gradient . Western blot against anti - V5 is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Fractionation of HeLa cells overexpressing either HSP27 or the P182L mutant by a 10% to 80% sucrose gradient. Western blot against anti-V5 is shown.


( C ) Expression of V5 - epitope - tagged HSP27 ( WT or P182L mutant ) in HSP27 knock - out HeLa cells and immunostaining of HSP27 . The scale bars correspond to the non - expanded dimensions , derived by dividing by the sample expansion factor of 4 . 3 . ( D ) The P182L mutation decreases the density of HSP27 spots upon detection of local fluorescence intensity maxima and quantification of the number of detected spots in expanded samples ( n = 30 ) . The error bar is the standard deviation for the different cytoplasmic regions . ( E ) Distribution of the intensity of individual spots ( n = 3342 ) after Gaussian fitting of fluorescence intensity in the neighborhood of detected spots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Expression of V5-epitope-tagged HSP27 (WT or P182L mutant) in HSP27 knock-out HeLa cells and immunostaining of HSP27. The scale bars correspond to the non-expanded dimensions, derived by dividing by the sample expansion factor of 4.3. (D) The P182L mutation decreases the density of HSP27 spots upon detection of local fluorescence intensity maxima and quantification of the number of detected spots in expanded samples (n = 30). The error bar is the standard deviation for the different cytoplasmic regions. (E) Distribution of the intensity of individual spots (n = 3342) after Gaussian fitting of fluorescence intensity in the neighborhood of detected spots.


( F ) Immunostaining of HSP27 ( white ) in motor neurons differentiated from CMT patient - derived iPSCs that harbor the heterozygous P182L mutation reveals large , cytoplasmic aggregates ( center and right panels ) . The control motor neurons that contain WT HSP27 do not show evidence of any aggregates . The nucleus is stained with Hoechst ( blue ) . Scale bar , 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Immunostaining of HSP27 (white) in motor neurons differentiated from CMT patient-derived iPSCs that harbor the heterozygous P182L mutation reveals large, cytoplasmic aggregates (center and right panels). The control motor neurons that contain WT HSP27 do not show evidence of any aggregates. The nucleus is stained with Hoechst (blue). Scale bar, 10 µm.


( A ) Primary sequences of the WT peptide ( red ) and P182L peptide ( blue ) . The IxI / V motif is indicated with white text . Below : regions of 2D 1H - 15N HSQC spectra of 15N - labeled cHSP27 ( black ) in the presence of increasing amounts of WT ( red ) or P182L peptide ( blue ) . Resonances in the β4 and β8 strands broaden and disappear upon peptide binding , indicative of intermediate exchange . ( B ) Zoomed - in regions of resonances during the peptide titrations . The color bars indicate the amount of added WT or P182L peptide . ( C ) The combined 1HN and 15N chemical shift changes , denoted as chemical shift perturbation ( CSP ) , are plotted as a function of added peptide concentration . ( D ) CSPs shown as a function of residue number for the WT ( red ) and P182L peptide ( blue ) . ( E ) X - ray structure of cHSP27 bound to the WT peptide ( PDB : 4mjh ) , shown in white sticks , with the results from ( D ) plotted onto the structure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Primary sequences of the WT peptide (red) and P182L peptide (blue). The IxI/V motif is indicated with white text. Below: regions of 2D 1H-15N HSQC spectra of 15N-labeled cHSP27 (black) in the presence of increasing amounts of WT (red) or P182L peptide (blue). Resonances in the β4 and β8 strands broaden and disappear upon peptide binding, indicative of intermediate exchange. (B) Zoomed-in regions of resonances during the peptide titrations. The color bars indicate the amount of added WT or P182L peptide. (C) The combined 1HN and 15N chemical shift changes, denoted as chemical shift perturbation (CSP), are plotted as a function of added peptide concentration. (D) CSPs shown as a function of residue number for the WT (red) and P182L peptide (blue). (E) X-ray structure of cHSP27 bound to the WT peptide (PDB: 4mjh), shown in white sticks, with the results from (D) plotted onto the structure.


( A , B ) i . 15N CPMG RD data for residues in the β4 strand ( T113 ) and the β8 strand ( S154 ) in the absence of added peptide . CPMG data are shown for the same residues in the presence of ii . ca . 2 % cHSP27 - WT peptide complex or iii . ca . 2 % of the cHSP27 - P182L peptide complex . Uncertainties in R2 , eff values are derived from the standard deviations of fitted peak intensities obtained from duplicated νCPMG values . ( C ) i . The association ( kon ) and dissociation ( koff ) rates for WT ( red ) and P182L ( blue ) IxI / V peptide binding to the ACD , calculated directly from the CPMG RD data . Residues that are impacted by the presence of a small amount of peptide are indicated by red ( ii . , WT ) and blue ( iii . , P182L ) spheres . In both cases , residues in similar ACD regions show enhanced Rex , indicative of chemical exchange at similar sites .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) i. 15N CPMG RD data for residues in the β4 strand (T113) and the β8 strand (S154) in the absence of added peptide. CPMG data are shown for the same residues in the presence of ii. ca. 2% cHSP27-WT peptide complex or iii. ca. 2% of the cHSP27-P182L peptide complex. Uncertainties in R2,eff values are derived from the standard deviations of fitted peak intensities obtained from duplicated νCPMG values. (C) i. The association (kon) and dissociation (koff) rates for WT (red) and P182L (blue) IxI/V peptide binding to the ACD, calculated directly from the CPMG RD data. Residues that are impacted by the presence of a small amount of peptide are indicated by red (ii., WT) and blue (iii., P182L) spheres. In both cases, residues in similar ACD regions show enhanced Rex, indicative of chemical exchange at similar sites.


( B ) Volcano plot representing the interactors that are enriched for the P182L mutant versus GFP as a negative control , obtained with affinity - enrichment mass spectrometry . Proteins that co - immunoprecipitated significantly more ( p < 0 . 05 ) with the P182L variant are shown in the upper right quadrant . Interactors with one or more [ I / V ] - x - [ I / V ] motifs are displayed in red while other significantly enriched interactors are displayed in black .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Volcano plot representing the interactors that are enriched for the P182L mutant versus GFP as a negative control, obtained with affinity-enrichment mass spectrometry. Proteins that co-immunoprecipitated significantly more (p<0.05) with the P182L variant are shown in the upper right quadrant. Interactors with one or more [I/V]-x-[I/V] motifs are displayed in red while other significantly enriched interactors are displayed in black.


( C - D ) Co - immunoprecipitation from HeLa cells stably overexpressing V5 - epitope - tagged HSP27 ( WT or P182L mutant ) using anti - V5 beads . Co - immunoprecipitation was quantified and presented as a percentage relative to the P182L variant ( n = 3 ) . The mean ± one standard deviation ( n = 3 ) is shown . Right : the location of Pro182 ( red ) in the crystal structure of peptide - bound cHSP27 ( PDB : 4mjh ) and in silico creation of the P182L mutation ( blue ) . The peptide is shown in white sticks . P182L specifically weakens binding of the HSP27 IxI / V motif .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) using anti-V5 beads. Co-immunoprecipitation was quantified and presented as a percentage relative to the P182L variant (n=3). The mean ± one standard deviation (n=3) is shown. Right: the location of Pro182 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the P182L mutation (blue). The peptide is shown in white sticks. P182L specifically weakens binding of the HSP27 IxI/V motif.


( E ) Co - immunoprecipitation from HeLa cells stably overexpressing V5 - epitope - tagged HSP27 ( WT or P182L mutant ) and transiently transfected with BAG3 - eGFP ( WT or IPV - mutant ) using anti - GFP beads . Western blots for anti - GFP ( BAG3 ) , V5 ( HSPB1 ) , and α - tubulin . NS indicates a non - specific band . Note that detection of BAG3 ( anti - GFP ) and HSP27 ( anti - V5 ) occurs with different antibodies . The locations of the IPV - to - GPG mutations in BAG3 are depicted below .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) and transiently transfected with BAG3-eGFP (WT or IPV-mutant) using anti-GFP beads. Western blots for anti-GFP (BAG3), V5 (HSPB1), and α-tubulin. NS indicates a non-specific band. Note that detection of BAG3 (anti-GFP) and HSP27 (anti-V5) occurs with different antibodies. The locations of the IPV-to-GPG mutations in BAG3 are depicted below.


( F ) Co - immunoprecipitation from HeLa cells transiently transfected with V5 - epitope - tagged HSP27 ( P182L or S155Q / P182L mutant ) using anti - V5 beads ( n = 2 ) . Right : the location of Ser155 ( red ) in the crystal structure of peptide - bound cHSP27 ( PDB : 4mjh ) and in silico creation of the S155Q mutation ( purple ) showing a clash with the bound peptide . The peptide is shown in white sticks . S155Q disrupts binding of all IxI / V motifs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Co-immunoprecipitation from HeLa cells transiently transfected with V5-epitope-tagged HSP27 (P182L or S155Q/P182L mutant) using anti-V5 beads (n=2). Right: the location of Ser155 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the S155Q mutation (purple) showing a clash with the bound peptide. The peptide is shown in white sticks.S155Q disrupts binding of all IxI/V motifs.


B ) Western blot assays for the expression level of TFAM in ten HCC cell lines , one normal liver cell line ( LO2 ) and two non - tumor liver tissues . The protein level was quantified as ratios between TFAM and GAPDH ( n = 3 independent experiments ) . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Western blot assays for the expression level of TFAM in ten HCC cell lines, one normal liver cell line (LO2) and two non-tumor liver tissues. The protein level was quantified as ratios between TFAM and GAPDH (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( C ) Western blot and quantitative analysis for the expression of TFAM in HCC cells with TFAM stable knockdown and control cells ( n = 3 independent experiments ) . shCtrl , control shRNA ; shTFAM , shRNA against TFAM ; EV , empty vector ; TFAM , expression vector encoding TFAM . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Western blot and quantitative analysis for the expression of TFAM in HCC cells with TFAM stable knockdown and control cells (n = 3 independent experiments). shCtrl, control shRNA; shTFAM, shRNA against TFAM; EV, empty vector; TFAM, expression vector encoding TFAM. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( D , E ) Wound - healing migration assay ( D ) and Transwell migration and invasion assays ( E ) for HCC cells with treatment as indicated ( n = 3 independent experiments ) . Scale bars , 100μm . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Wound-healing migration assay (D) and Transwell migration and invasion assays (E) for HCC cells with treatment as indicated (n = 3 independent experiments). Scale bars, 100μm. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( G ) Representative H & E images of lung metastasis in mice used in ( F ) . Scale bars , 100μm . ( H , Number of metastatic nodules per lung ( H ) in mice used in ( F ) . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * * P < 0 . 01 .

(G) Representative H&E images of lung metastasis in mice used in (F). Scale bars, 100μm. (H, Number of metastatic nodules per lung (H) in mice used in (F). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( C ) Immunofluorescence analysis of TFAM and Phalloidin staining in human HCC cells from paired clinical samples ( n = 100 ) . The 3D reconstruction of z - stacks from nuclear region is showed in the right side . Red : LifeAct , Blue : DAPI , Green : TFAM . Scale bars , 20μm for microscopy images , 40μm for 3D reconstruction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Immunofluorescence analysis of TFAM and Phalloidin staining in human HCC cells from paired clinical samples (n=100). The 3D reconstruction of z-stacks from nuclear region is showed in the right side. Red: LifeAct, Blue: DAPI, Green: TFAM. Scale bars, 20μm for microscopy images, 40μm for 3D reconstruction.


( D ) Statistical analysis for fluorescent intensity of nuclear F - actin in ( A ) , ( B ) and ( C ) . Fluorescence intensity are quantified from at least 30 cells for each group in ( A ) and ( B ) . One - way ANOVA . * P < 0 . 05 ; * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Statistical analysis for fluorescent intensity of nuclear F-actin in (A), (B) and (C). Fluorescence intensity are quantified from at least 30 cells for each group in (A) and (B). One-way ANOVA. *P < 0.05; **P < 0.01.


( E ) Representative H & E images of lung metastasis in mice used in ( D ) . Scale bars , 100μm . ( F and G ) Number of metastatic nodules per lung in mice used in ( F ) and the incidence of lung metastasis ( G ) in mice used in ( D ) . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Representative H&E images of lung metastasis in mice used in (D). Scale bars, 100μm. (F and G) Number of metastatic nodules per lung in mice used in (F) and the incidence of lung metastasis (G) in mice used in (D). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


F ) Western blot assays for protein expression levels of metastasis related gene in HCC cells ( n = 3 independent experiments ) and tumor tissues from mice ( n = 4 per group ) with treatments as indicated . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Western blot assays for protein expression levels of metastasis related gene in HCC cells (n = 3 independent experiments) and tumor tissues from mice (n =4 per group) with treatments as indicated. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. *P < 0.05; **P < 0.01.


( H Western blot assays levels of FN1 , ITGB3 in HCC cells with treatment as indicated ( n = 3 independent experiments ) . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 .

(H Western blot assays levels of FN1, ITGB3 in HCC cells with treatment as indicated (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. *P < 0.05; **P < 0.01.


( A ) Western blot analyses for the protein level of key regulators of actin polymerization in the cytoplasm and nucleus of HCC cells with treatment as indicated . Tublin and Lamin B1 were used as loading controls for the cytoplasm and nucleus , respectively ( n = 3 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Western blot analyses for the protein level of key regulators of actin polymerization in the cytoplasm and nucleus of HCC cells with treatment as indicated. Tublin and Lamin B1 were used as loading controls for the cytoplasm and nucleus, respectively (n = 3 independent experiments).


( B ) Western blot analyses for the protein level in tissues of mouse liver tumor induced by Akt / β - catenin ( n = 3 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Western blot analyses for the protein level in tissues of mouse liver tumor induced by Akt/β-catenin (n = 3 independent experiments).


Representative IHC images of mDia2 nuclear translocation in human ( E ) HCC tissues . NT , non - tumor liver tissues , n = 100 ; MT , metastatic tumor tissues , n = 58 ; NMT , non - metastatic tumor tissues n = 42 . Scale bars , 25μm . ( F ) Positive rates of mDia2 in nucleus and cytoplasm was calculated based on the IHC assay in ( E ) . Graphs show mean ± s . e . m . One - way ANOVA . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative IHC images of mDia2 nuclear translocation in human (E) HCC tissues. NT, non-tumor liver tissues, n=100; MT, metastatic tumor tissues, n=58; NMT, non-metastatic tumor tissues n=42. Scale bars, 25μm. (F) Positive rates of mDia2 in nucleus and cytoplasm was calculated based on the IHC assay in (E). Graphs show mean ± s.e.m. One-way ANOVA. **P < 0.01.


( H and I ) Spearman correlation analysis between the positive rates of nuclear mDia2 and the TFAM expression level ( or positive rates of nuclear F - actin ) based on the IHC assay in ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H and I) Spearman correlation analysis between the positive rates of nuclear mDia2 and the TFAM expression level (or positive rates of nuclear F-actin) based on the IHC assay in (E).


( D ) Western blot analysis of anti - mDia2 IPs and whole - cell lysates ( WCL ) derived from HCC cells and mouse tumor tissues ( n = 3 independent experiments ) . NT , non - tumor liver tissues ; MT , metastatic tumor tissues ; NMT , non - metastatic tumor tissues .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Western blot analysis of anti-mDia2 IPs and whole-cell lysates (WCL) derived from HCC cells and mouse tumor tissues (n = 3 independent experiments). NT, non-tumor liver tissues; MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues.


( E ) The level of malonylated mDia2 . Equal amount of immunoprecipitated mDia2 either from TFAM knockdown or control cells was loaded in Western blot assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) The level of malonylated mDia2. Equal amount of immunoprecipitated mDia2 either from TFAM knockdown or control cells was loaded in Western blot assay.


( F ) Western blot analysis for the distribution of mDia2 in SNU - 368 cells incubated with malonyl - CoA ( 5 μM ) or ND - 630 ( 10 μM ) ( n = 3 independent experiments ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

(F) Western blot analysis for the distribution of mDia2 in SNU-368 cells incubated with malonyl-CoA (5 μM) or ND-630 (10 μM) (n = 3 independent experiments).


( H ) Representative H & E images of lung metastasis in mice used in ( G ) . Scale bars , 100μm . ( I ) Number of metastatic nodules per lung in mice used in ( G ) . Data information : Graphs show mean ± s . e . m . , two - tailed unpaired t - test for , I ) . * P < 0 . 05 ; * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative H&E images of lung metastasis in mice used in (G). Scale bars, 100μm. (I) Number of metastatic nodules per lung in mice used in (G). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test for , I). *P < 0.05; **P < 0.01.


( A ) IHC scores of TFAM in 429 paired tissues from HCC patients . The relative expression ratio of tumor to non - tumor was log2 - transformed . T , tumor tissues ; NT , non - tumor liver tissues .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) IHC scores of TFAM in 429 paired tissues from HCC patients. The relative expression ratio of tumor to non-tumor was log2-transformed. T, tumor tissues; NT, non-tumor liver tissues.


( B ) mRNA level of TFAM in 50 HCC patients ' paired tissues from TCGA database . The relative expression ratio of tumor to non - tumor was log2 - transformed .

(B) mRNA level of TFAM in 50 HCC patients' paired tissues from TCGA database. The relative expression ratio of tumor to non-tumor was log2-transformed.


( C ) Representative images of IHC staining of TFAM ( top panel ) and scores ( bottom panel ) in HCC tumor tissues . One - way ANOVA . * P < 0 . 05 ; * * P < 0 . 01 . MT , metastatic tumor tissues ; NMT , non - metastatic tumor tissues . Scale bars , 25μm . Boxes : first quartile to third quartile ; Whiskers : minimum value to maximum value ; Central band : median .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative images of IHC staining of TFAM (top panel) and scores (bottom panel) in HCC tumor tissues. One-way ANOVA. *P < 0.05; **P < 0.01. MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues. Scale bars, 25μm. Boxes: first quartile to third quartile; Whiskers: minimum value to maximum value; Central band: median.


( D Kaplan - Meier curves for overall survival of HCC patients stratified by TFAM expression . ( F Kaplan - Meier curves for overall survival of HCC patients stratified by nuclear F - actin positive rates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D Kaplan-Meier curves for overall survival of HCC patients stratified by TFAM expression. (F Kaplan-Meier curves for overall survival of HCC patients stratified by nuclear F-actin positive rates.


E ) the recurrence of HCC patients stratified by TFAM expression . G ) the recurrence of HCC patients stratified by nuclear F - actin positive rates .

E) the recurrence of HCC patients stratified by TFAM expression. G) the recurrence of HCC patients stratified by nuclear F-actin positive rates.


( H ) Representative images of IHC staining of TFAM , FN1 and ITGB3 from two HCC patients . Scale bars , 25μm . ( I ) Spearman correlation analysis between the levels of TFAM and FN1 , IL - 6 , TGFB1 or ITGB3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative images of IHC staining of TFAM, FN1 and ITGB3 from two HCC patients. Scale bars, 25μm. (I) Spearman correlation analysis between the levels of TFAM and FN1, IL-6, TGFB1 or ITGB3.


b , Representative confocal images of NeuN ( green ) and Npas4 ( red ) immunostaining performed 1h after the last injection of cocaine or saline ( scale bar : 50 µm ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

b, Representative confocal images of NeuN (green) and Npas4 (red) immunostaining performed 1h after the last injection of cocaine or saline (scale bar: 50 µm).


c , Quantifications of Npas4 - positive cells ( fold change normalized to the 30min saline control group ) in the NAc and dStr . Mean + SEM ; n = 4 mice per group . 10 images ( 5 per hemisphere ) per mouse for the NAc and 10 images ( 5 per hemisphere ) for the dStr . p * * < 0 . 01 , unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

c, Quantifications of Npas4-positive cells (fold change normalized to the 30min saline control group) in the NAc and dStr. Mean + SEM; n = 4 mice per group. 10 images (5 per hemisphere) per mouse for the NAc and 10 images (5 per hemisphere) for the dStr. p** < 0.01, unpaired Student's t-test.


c , MSN cultures at DIV11 were treated for 2h with either 10 µM of the D1 - agonist SKF - 38393 ( SKF ) alone or with SKF and 10 µM of the selective D1 - antagonist SCH - 23390 ( SCH ) . qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos and Arc . Mean + SEM ; one - way ANOVA with Holm - Sidak post - hoc correction ; p * * < 0 . 01 , p * * * * < 0 . 0001 ; n = 3 independent cultures as biological replicates .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

c, MSN cultures at DIV11 were treated for 2h with either 10 µM of the D1-agonist SKF-38393 (SKF) alone or with SKF and 10 µM of the selective D1-antagonist SCH-23390 (SCH). qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos and Arc. Mean + SEM; one-way ANOVA with Holm-Sidak post-hoc correction; p** < 0.01, p**** < 0.0001; n = 3 independent cultures as biological replicates.


d , MSNs were depolarized with potassium chloride ( HiK , 55 mM ) or treated with the D1 - agonist SKF - 38393 ( 10 µM ) for 1h , 2h or 4h . Subsequently , qPCR was performed to assess immediate early gene mRNA levels . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test ; indicated p - values for induction over control for each treatment ; p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 ; n = 4 independent cultures as biological replicates .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

d, MSNs were depolarized with potassium chloride (HiK, 55 mM) or treated with the D1-agonist SKF-38393 (10 µM) for 1h, 2h or 4h. Subsequently, qPCR was performed to assess immediate early gene mRNA levels. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test; indicated p-values for induction over control for each treatment; p* < 0.05, p** < 0.01, p***<0.001; n = 4 independent cultures as biological replicates.


g , Differential induction of Npas4 through HiK and not forskolin in hiPSC - derived forebrain organoids . Organoids were treated with HiK ( 55 mM ) or the PKA activator forskolin plus TTX ( Fsk , 10 µM ; TTX 1 µM ) for 1h before qPCR analysis was performed to assess mRNA levels of Npas4 and Arc . Mean + SEM ; n = 7 organoids in control condition , n = 5 organoids for HiK , n = 5 organoids for Fsk / TTX . One - way ANOVA with Tukey ' s post - hoc test , ns = not significant , p * < 0 . 05 , p * * * < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

g, Differential induction of Npas4 through HiK and not forskolin in hiPSC-derived forebrain organoids. Organoids were treated with HiK (55 mM) or the PKA activator forskolin plus TTX (Fsk, 10 µM; TTX 1 µM) for 1h before qPCR analysis was performed to assess mRNA levels of Npas4 and Arc. Mean + SEM; n = 7 organoids in control condition, n = 5 organoids for HiK, n = 5 organoids for Fsk/TTX. One-way ANOVA with Tukey's post-hoc test, ns = not significant, p* < 0.05, p***<0.001.


MSN cultures were stimulated with HiK ( 55 mM ) for 1h in the presence of various pharmacologicals to dissect the synapse - to - nucleus cascades mediating transcription of Npas4 , Fosb and Arc . Values were normalized to the induced condition to compare for differences in induction between treatments . Mean + SEM and one - way ANOVA with Bonferroni post - hoc correction were used in all experiments . p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 , p * * * * < 0 . 0001 . n - number indicated in each panel . a , co - incubation with the NMDA receptor blocker APV ( 100 mM , applied 5min before ) reveals that all IEG inductions through HiK in MSNs depend on NDMA receptor activity . Mean + SEM ; n = 4 independent cultures as biological replicates . b , blockade of voltage - dependent calcium channels through verapamil ( 30 µM , 5min before ) and TTX ( 1 µM , 5min , to prevent verapamil - induced bursting ) leads to significant reduction in gene - inductions for Npas4 and Fosb . Mean + SEM ; n = 4 independent cultures as biological replicates . c , inhibition of ERK1 / 2 through UO126 ( 10 µM , 20min before ) leads to significant blockade of mRNA induction for Arc and Fosb , but not Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates . d , inhibition of p38alpha and p38beta through SB203580 or SB202190 respectively ( both 10 µM and 20min before ) leads to significant reductions in mRNA induction for Arc and Fosb , but not Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates . e , blockade of PKA activity through H - 89 ( 10 µM , 20min before ) leads to reduced induction of Arc , unaffected induction of Fosb and a superinduction of Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates . f , inhibition of calcineurin through co - application of Cyclosporin and FK506 ( 1 µM each , 20min before ) leads to unaffected induction of mRNAs for Arc and Fosb , but significantly reduced levels of Npas4 . Mean + SEM ; n = 3 independent cultures as biological replicates .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of Npas4, Fosb and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. a, co-incubation with the NMDA receptor blocker APV (100 mM, applied 5min before) reveals that all IEG inductions through HiK in MSNs depend on NDMA receptor activity. Mean + SEM; n = 4 independent cultures as biological replicates. b, blockade of voltage-dependent calcium channels through verapamil (30 µM, 5min before) and TTX (1 µM, 5min, to prevent verapamil-induced bursting) leads to significant reduction in gene-inductions for Npas4 and Fosb. Mean + SEM; n =4 independent cultures as biological replicates. c, inhibition of ERK1/2 through UO126 (10 µM, 20min before) leads to significant blockade of mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. d, inhibition of p38alpha and p38beta through SB203580 or SB202190 respectively (both 10 µM and 20min before) leads to significant reductions in mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. e, blockade of PKA activity through H-89 (10 µM, 20min before) leads to reduced induction of Arc, unaffected induction of Fosb and a superinduction of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. f, inhibition of calcineurin through co-application of Cyclosporin and FK506 (1 µM each, 20min before) leads to unaffected induction of mRNAs for Arc and Fosb, but significantly reduced levels of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates.


MSN cultures were stimulated with HiK ( 55 mM ) for 1h in the presence of various pharmacologicals to dissect the synapse - to - nucleus cascades mediating transcription of Npas4 , Fosb and Arc . Values were normalized to the induced condition to compare for differences in induction between treatments . Mean + SEM and one - way ANOVA with Bonferroni post - hoc correction were used in all experiments . p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 , p * * * * < 0 . 0001 . n - number indicated in each panel . g , blockade of CaMKII / IV through application of KN - 93 ( 1 µM , 20min before ) leads to significantly reduced mRNA inductions of Arc , Fosb and Npas4 . Mean + SEM ; n = 4 independent cultures as biological replicates . h , co - application of HiK and SKF reveals synergistic effects on mRNA induction of Fosb but not Arc or Npas4 , Mean + SEM ; n = 6 independent cultures as biological replicates .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of Npas4, Fosb and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. g, blockade of CaMKII/IV through application of KN-93 (1 µM, 20min before) leads to significantly reduced mRNA inductions of Arc, Fosb and Npas4. Mean + SEM; n = 4 independent cultures as biological replicates. h, co-application of HiK and SKF reveals synergistic effects on mRNA induction of Fosb but not Arc or Npas4, Mean + SEM; n = 6 independent cultures as biological replicates.


investigating the role of nuclear calcium signaling in mRNA induction of Arc , Fosb and Npas4 . k , immunohistochemical analysis of MSNs infected with mCherry - NLS as control or the competitive nuclear Ca2 + / CaM antagonist CaMBP4 - mCherry . Infection on DIV4 , fixation on DIV11 . D32 = anti - Darpp - 32 . scale bar = 10 µm . l , MSNs expressing rAAV - mCherry - control or CaMBP4 ( BP4 ) were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test , ns = not significant , p * * * * < 0 . 0001 . n = 5 independent cultures as biological replicates . m , immunohistochemical analysis of MSNs infected with mCherry - NLS as control or the nuclear Ca2 + - buffer Parvalbumin - NLS - mCherry . Infection on DIV4 , fixation on DIV11 . D32 = anti - Darpp - 32 . n , MSNs expressing rAAV - mCherry - control or PV - NLS were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test , p * < 0 . 05 , p * * < 0 . 01 , p * * * * < 0 . 0001 . n = 3 independent cultures as biological replicates .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

investigating the role of nuclear calcium signaling in mRNA induction of Arc, Fosb and Npas4. k, immunohistochemical analysis of MSNs infected with mCherry-NLS as control or the competitive nuclear Ca2+/CaM antagonist CaMBP4-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. scale bar = 10 µm. l, MSNs expressing rAAV-mCherry-control or CaMBP4 (BP4) were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, ns = not significant, p****<0.0001. n = 5 independent cultures as biological replicates. m, immunohistochemical analysis of MSNs infected with mCherry-NLS as control or the nuclear Ca2+-buffer Parvalbumin-NLS-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. n, MSNs expressing rAAV-mCherry-control or PV-NLS were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p** < 0.01, p****<0.0001. n = 3 independent cultures as biological replicates.


e , Npas4 controls activity - dependent upregulation of genes for glutamatergic synapse formation and Ca2 + channels . MSNs were infected with rAAVs expressing shRNA - scrambled ( control ) or shNpas4 ( Npas4 knockdown ) on DIV5 . On DIV 12 , cultures were stimulated with HiK ( 55 mM ) for 1h or 6h and mRNA levels were analysed by qPCR . mRNA level = fold change in mRNA compared to shscrambled unstimulated control . Mean + SEM ; two - way ANOVA with Bonferroni post - hoc test , p * < 0 . 05 , p * * * < 0 . 001 . n = 5 independent cultures as biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

e, Npas4 controls activity-dependent upregulation of genes for glutamatergic synapse formation and Ca2+ channels. MSNs were infected with rAAVs expressing shRNA-scrambled (control) or shNpas4 (Npas4 knockdown) on DIV5. On DIV 12, cultures were stimulated with HiK (55 mM) for 1h or 6h and mRNA levels were analysed by qPCR. mRNA level = fold change in mRNA compared to shscrambled unstimulated control. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p***<0.001. n = 5 independent cultures as biological replicates.


e , Confocal images of tdTomato - labelled D1 - MSNs expressing shscr - GFP or shNpas4 - GFP ( left panels ; scale bar : 20 µm ) . 3D volume - renderings of MSN dendrites from the NAc shell ( right panels ; scale bar : 2 µm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

e, Confocal images of tdTomato-labelled D1-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm).


g , Confocal images of tdTomato - labelled D2 - MSNs expressing shscr - GFP or shNpas4 - GFP ( left panels ; scale bar : 20 µm ) . 3D volume - renderings of MSN dendrites from the NAc shell ( right panels ; scale bar : 2 µm ) .

g, Confocal images of tdTomato-labelled D2-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm).