Figure 9A Colocalization of WIPI1 with Vps26A . The indicated WIPI1mCherry variants and VPS26EGFP were expressed for 18 h in HK2 cells , from which endogenous WIPI1 had been deleted ( WIPI1 - KO ) . The cells were analyzed by confocal microscopy . Scale bars : 10 μm . Insets show enlargements of the outlined areas . B Colocalization between WIPI1 variants and Vps26 was quantified in the cells from A using the Manders ' colocalization coefficient M2 . The red line indicates the mean ; n = 120 cells per condition , pooled from three independent experiments . P - values were calculated by t - test ( analysis performed with 99 % confidence * * * p < 0 . 0001 ) . C Expression levels of WIPI1mCherry variants . Lysates ( 50 μg of protein per sample ) from the cells in A were analyzed by SDS - PAGE and Western blot against WIPI1 and tubulin . D Depletion of VPS35 . HK2 cells expressing the indicated WIPI1eGFP variants were transfected with siRNA against VPS35 ( VPS35 KD ) or a control siRNA pool . Lysates ( 50 μg per sample ) from the cells were analyzed by SDS - PAGE and Western blot against Vps35 and tubulin . E Blots from D were quantified on a LICOR Odyssey fluorescence imager . n = 3 . Red lines indicate the means , n = 3 independent experiments , using a Welch ' s t - test statistical analysis . Bars represent the mean and error bars the SD . F Cells from D were analyzed by confocal microscopy 18 h after transfection . Scale bar : 10 μm . G Quantification of tubule length in the cells from F . Data are means ± SD . of n = 210 tubules per condition , from three independent experiments . p values were calculated by unpaired Student ' s t - test . 99 % confidence : * * * = p < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9A Colocalization of WIPI1 with Vps26A. The indicated WIPI1mCherry variants and VPS26EGFP were expressed for 18 h in HK2 cells, from which endogenous WIPI1 had been deleted (WIPI1-KO). The cells were analyzed by confocal microscopy. Scale bars: 10 μm. Insets show enlargements of the outlined areas. B Colocalization between WIPI1 variants and Vps26 was quantified in the cells from A using the Manders' colocalization coefficient M2. The red line indicates the mean; n = 120 cells per condition, pooled from three independent experiments. P-values were calculated by t-test (analysis performed with 99% confidence ***p < 0.0001). C Expression levels of WIPI1mCherry variants. Lysates (50 μg of protein per sample) from the cells in A were analyzed by SDS-PAGE and Western blot against WIPI1 and tubulin.D Depletion of VPS35. HK2 cells expressing the indicated WIPI1eGFP variants were transfected with siRNA against VPS35 (VPS35 KD) or a control siRNA pool. Lysates (50 μg per sample) from the cells were analyzed by SDS-PAGE and Western blot against Vps35 and tubulin. E Blots from D were quantified on a LICOR Odyssey fluorescence imager. n=3. Red lines indicate the means, n = 3 independent experiments, using a Welch's t-test statistical analysis. Bars represent the mean and error bars the SD.F Cells from D were analyzed by confocal microscopy 18 h after transfection. Scale bar: 10 μm. G Quantification of tubule length in the cells from F. Data are means ± SD. of n=210 tubules per condition, from three independent experiments. p values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.


Figure 10A , Tf recycling . WIPI1 - KO cells were transfected with WIPI1WT - eGFP , WIPI1S69E - eGFP or WIPI1S69A - eGFP for 18h . Then , they were serum - starved for 1 h , loaded with Alexa Fluor 568 - conjugated Tf , chased at 37 ° C for 1 h without labeled Tf , and analyzed by confocal microscopy . Scale bar : 10 μm . White dashed lines delineate the circumference of the cells . B , Quantification of Tf - fluorescence in the cells from a that expressed WIPI1 variants . Total cell fluorescence was integrated and corrected for background fluorescence . Mean values ± SD are shown . n = 3 independent experiments with a total of 150 cells analyzed per condition . P values were calculated by unpaired Student ' s t - test . 99 % confidence : * * * = p < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 10A, Tf recycling. WIPI1-KO cells were transfected with WIPI1WT-eGFP, WIPI1S69E-eGFP or WIPI1S69A-eGFP for 18h. Then, they were serum-starved for 1 h, loaded with Alexa Fluor 568-conjugated Tf, chased at 37°C for 1 h without labeled Tf, and analyzed by confocal microscopy. Scale bar: 10 μm. White dashed lines delineate the circumference of the cells. B, Quantification of Tf-fluorescence in the cells from a that expressed WIPI1 variants. Total cell fluorescence was integrated and corrected for background fluorescence. Mean values ± SD are shown. n = 3 independent experiments with a total of 150 cells analyzed per condition. P values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.


Figure 1A . Confocal microscopy analysis showing the localization of endogenous Rab22a ( green ) and different endocytic markers ( red ) : early endosomal marker EEA1 , late endosome / lysosomal marker Lamp1 and the recycling compartment marker TfR in BMDCs at steady state . The means ± SEM of the Pearson correlation coefficients ( Pcc ) from Rab22a and EEA1 ( 0 . 4317 ± 0 . 05101 ) , Lamp1 ( 0 . 8412 ± 0 . 03562 ) and TfR ( 0 . 6409 ± 0 . 02999 ) were estimated from 15 images analyzed for each marker . Scale bars : 5 µm . B . Colocalization of endogenous Rab22a ( red ) and EEA1 ( magenta ) around endosomes containing fluorescent soluble OVA ( OVA - FITC , green ) after 30 min of internalization by BMDCs . More than 60 % of the endosomes were positive for Rab22a and EEA1 . The indicated boxes are shown at higher magnification in the insets . Scale bars : 5 µm . C . IF detection of endogenous Rab22a ( green ) and Lamp1 ( red ) after 1 h of phagocytosis of 3 µm latex beads ( LB ) in BMDCs . Around 50 % of the phagosomes were double positive for Rab22a and Lamp1 . Asterisks indicate the LB . Scale bars : 5 µm . D . BMDCs were incubated with 3 μm magnetic beads for 15 min at 37 ° C and chased for 0 or 45 min . The panel shows immunoblotting of purified phagosomes and the total cell lysates ( TCL ) analyzed for Lamp1 and Rab22a . A total protein amount of 10 µg and 50 µg was loaded for purified phagosomes and TCL , respectively . The blot is representative of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Confocal microscopy analysis showing the localization of endogenous Rab22a (green) and different endocytic markers (red): early endosomal marker EEA1, late endosome/lysosomal marker Lamp1 and the recycling compartment marker TfR in BMDCs at steady state. The means ± SEM of the Pearson correlation coefficients (Pcc) from Rab22a and EEA1 (0.4317 ± 0.05101), Lamp1 (0.8412 ± 0.03562) and TfR (0.6409 ± 0.02999) were estimated from 15 images analyzed for each marker. Scale bars: 5 µm.B. Colocalization of endogenous Rab22a (red) and EEA1 (magenta) around endosomes containing fluorescent soluble OVA (OVA-FITC, green) after 30 min of internalization by BMDCs. More than 60% of the endosomes were positive for Rab22a and EEA1. The indicated boxes are shown at higher magnification in the insets. Scale bars: 5 µm.C. IF detection of endogenous Rab22a (green) and Lamp1 (red) after 1 h of phagocytosis of 3 µm latex beads (LB) in BMDCs. Around 50% of the phagosomes were double positive for Rab22a and Lamp1. Asterisks indicate the LB. Scale bars: 5 µm.D. BMDCs were incubated with 3 μm magnetic beads for 15 min at 37°C and chased for 0 or 45 min. The panel shows immunoblotting of purified phagosomes and the total cell lysates (TCL) analyzed for Lamp1 and Rab22a. A total protein amount of 10 µg and 50 µg was loaded for purified phagosomes and TCL, respectively. The blot is representative of three independent experiments.


Figure 2A . Immunoblotting and densitometry quantification of Rab22a in JAWS - IIDCs infected with lentiviruses encoding a random sequence ( Scramble ) and a shRNA specific for silencing Rab22a ( Rab22a KD ) . Data are representative of at least three independent experiments . B . RNA extraction and qPCR quantification of Rab22a in the same cells analyzed in A . Data show mean ± SEM of triplicate values and are representative of two independent experiments . * * P = 0 . 0047 . C . IF labeling and confocal microscopy analysis showing the distribution of MHC - I molecules ( H - 2Kb , green ) and Rab22a ( red ) in Scramble and Rab22a KD JAWS - II DCs . Nuclei stained with DAPI and DIC images are shown on the left . Overlay is shown in the right panels . Scale bars : 5 µm . Data are representative of at least 30 images analyzed for each experimental condition from three independent experiments . D . FACS analysis of MHC - I labeled in intact ( cell surface ) and permeabilized ( total ) Scramble and Rab22a KD JAWS - II cells . Data represent mean ± SEM of triplicate values and are representative of three independent experiments . P = 0 . 1082 ( ns ) and * * * P = 0 . 0003 . E . MHC - I staining on isolated phagosomes was measured by FACS at the indicated time periods after 3 µm LB internalization by Scramble and Rab22a KD JAWS - II DCs . Data represent mean ± SEM of three independent experiments . * * * P < 0 . 001 at 3 h and * P < 0 . 05 at 5 h between both DC types . F . MHC - I molecules recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS - II DCs . Data represent mean ± SEM of three independent experiments . * P < 0 . 05 at 10 min and * * P < 0 . 01 at 20 and 40 min . G . The transferrin ( Tfn ) recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS - II DCs . Data represent mean ± SEM of three independent experiments . P > 0 . 05 ( ns ) at 10 , 20 and 40 min .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Immunoblotting and densitometry quantification of Rab22a in JAWS-IIDCs infected with lentiviruses encoding a random sequence (Scramble) and a shRNA specific for silencing Rab22a (Rab22a KD). Data are representative of at least three independent experiments.B. RNA extraction and qPCR quantification of Rab22a in the same cells analyzed in A. Data show mean ± SEM of triplicate values and are representative of two independent experiments. ** P = 0.0047.C. IF labeling and confocal microscopy analysis showing the distribution of MHC-I molecules (H-2Kb, green) and Rab22a (red) in Scramble and Rab22a KD JAWS-II DCs. Nuclei stained with DAPI and DIC images are shown on the left. Overlay is shown in the right panels. Scale bars: 5 µm. Data are representative of at least 30 images analyzed for each experimental condition from three independent experiments.D. FACS analysis of MHC-I labeled in intact (cell surface) and permeabilized (total) Scramble and Rab22a KD JAWS-II cells. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. P = 0.1082 (ns) and ***P = 0.0003.E. MHC-I staining on isolated phagosomes was measured by FACS at the indicated time periods after 3 µm LB internalization by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. ***P < 0.001 at 3 h and *P < 0.05 at 5 h between both DC types.F. MHC-I molecules recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. *P < 0.05 at 10 min and **P < 0.01 at 20 and 40 min.G. The transferrin (Tfn) recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. P > 0.05 (ns) at 10, 20 and 40 min.


Figure 3A - C . The cross - presentation ability after incubation with A ) soluble OVA , B ) OVA / BSA - coated beads and C ) the SIINFEKL control peptide at the indicated concentrations by Scramble and Rab22a KD JAWS - II DCs was evaluated with the B3Z hybridoma . Data represent mean ± SEM of triplicate values and are representative of three independent experiments . A ) * * * P = 0 . 0001 and B ) P = 0 . 1432 ( ns ) ; * * P = 0 . 0044 . The two - tailed Student ' s unpaired t test was performed . D - E . Evaluation of endocytosis and phagocytosis in Scramble and Rab22a KD JAWS - II DCs . D ) The endocytosis of fluorescent OVA after 1 h of internalization and E ) the phagocytosis of 3 µm fluorescent LB at different times of internalization were assessed by FACS analysis . The antigen internalization was conducted at 37 ° C for effective uptake and at 4 ° C as negative control . Data represent mean ± SEM of triplicate values and are representative of three independent experiments . F . The kinetics of OVA degradation , as percentage of proteases inhibitors , in isolated phagosomes at the indicated time periods post - internalization from Scramble and Rab22a KD JAWS - II DCs was assessed by FACS analysis . Data represent mean ± SEM of three independent experiments . G . Immunoblotting of Rab22a and Actin in BMDCs infected with lentiviruses encoding a random sequence ( Scramble ) and two shRNA specific for silencing Rab22a ( Rab22a KD # 1 and # 2 ) . H - I . The cross - presentation capacity after the incubation with H ) soluble OVA at the indicated concentrations by Scramble , Rab22a KD # 1 and Rab22a KD # 2 BMDCs was evaluated as described before for JAWS - II DCs . Data represent mean ± SEM of triplicate values and are representative of two independent experiments . * * * P = 0 . 0001 . The two - tailed Student ' s unpaired t test was performed . H - I . The cross - presentation capacity after the incubation with I ) the SIINFEKL control peptide at the indicated concentrations by Scramble , Rab22a KD # 1 and Rab22a KD # 2 BMDCs was evaluated as described before for JAWS - II DCs . Data represent mean ± SEM of triplicate values and are representative of two independent experiments . * * * P = 0 . 0001 . The two - tailed Student ' s unpaired t test was performed . J . Soluble OVA was electroporated into the cytosol of Scramble and Rab22a KD JAWS - II DCs and T cell activation was determined 2 h later with the B3Z hibridoma . To control endogenous MHC - I antigen presentation specificity , DCs were also treated with brefreldin A ( BFA ) . The use of this drug markedly reduced CD8 + T cell response to similar levels obtained by DCs without any antigen ( ∅ ) . Data represent mean ± SEM of triplicate values and are representative of three independent experiments . K . Immunoblotting showing the amount of OVA incorporated by Scramble ( a ) and Rab22a KD ( b ) JAWS - II DCs after electroporation and BFA treatment .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-C. The cross-presentation ability after incubation with A) soluble OVA, B) OVA/BSA-coated beads and C) the SIINFEKL control peptide at the indicated concentrations by Scramble and Rab22a KD JAWS-II DCs was evaluated with the B3Z hybridoma. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. A) ***P = 0.0001 and B) P = 0.1432 (ns); **P = 0.0044. The two-tailed Student's unpaired t test was performed.D-E. Evaluation of endocytosis and phagocytosis in Scramble and Rab22a KD JAWS-II DCs. D) The endocytosis of fluorescent OVA after 1 h of internalization and E) the phagocytosis of 3 µm fluorescent LB at different times of internalization were assessed by FACS analysis. The antigen internalization was conducted at 37°C for effective uptake and at 4°C as negative control. Data represent mean ± SEM of triplicate values and are representative of three independent experiments.F. The kinetics of OVA degradation, as percentage of proteases inhibitors, in isolated phagosomes at the indicated time periods post-internalization from Scramble and Rab22a KD JAWS-II DCs was assessed by FACS analysis. Data represent mean ± SEM of three independent experiments.G. Immunoblotting of Rab22a and Actin in BMDCs infected with lentiviruses encoding a random sequence (Scramble) and two shRNA specific for silencing Rab22a (Rab22a KD #1 and #2).H-I. The cross-presentation capacity after the incubation with H) soluble OVA at the indicated concentrations by Scramble, Rab22a KD #1 and Rab22a KD #2 BMDCs was evaluated as described before for JAWS-II DCs. Data represent mean ± SEM of triplicate values and are representative of two independent experiments. ***P = 0.0001. The two-tailed Student's unpaired t test was performed.H-I. The cross-presentation capacity after the incubation with I) the SIINFEKL control peptide at the indicated concentrations by Scramble, Rab22a KD #1 and Rab22a KD #2 BMDCs was evaluated as described before for JAWS-II DCs. Data represent mean ± SEM of triplicate values and are representative of two independent experiments. ***P = 0.0001. The two-tailed Student's unpaired t test was performed.J. Soluble OVA was electroporated into the cytosol of Scramble and Rab22a KD JAWS-II DCs and T cell activation was determined 2 h later with the B3Z hibridoma. To control endogenous MHC-I antigen presentation specificity, DCs were also treated with brefreldin A (BFA). The use of this drug markedly reduced CD8+ T cell response to similar levels obtained by DCs without any antigen (∅). Data represent mean ± SEM of triplicate values and are representative of three independent experiments.K. Immunoblotting showing the amount of OVA incorporated by Scramble (a) and Rab22a KD (b) JAWS-II DCs after electroporation and BFA treatment.


Figure 4A . Scramble and Rab22a KD JAWS - II DCs were infected with OVA - YFP - expressing T . gondii ( TgRHYFPSAG1 - OVA ) for 8 h and confocal images detecting the parasite ( green ) , endogenous Rab22a ( red ) and GRA6 ( magenta ) were taken . Top panels : Scramble cells ; bottom panels : Rab22a KD cells . White boxes are shown at higher magnification in the insets . The nuclear marker DAPI ( blue ) and DIC images are shown in the left panels . Overlays are shown in the right panels . Scale bars : 5 µm . Images are representative of at least 30 analyzed from three independent experiments . B . The cross - presentation of OVA secreted by T . gondii ( TgRHYFPSAG1 - OVA ) after 8 h of infection at the indicated MOI was evaluated by B3Z T cell activation . Data represent mean ± SEM of triplicate values and are representative of three independent experiments . * * * P < 0 . 001 . A two - way ANOVA and the Bonferroni post - test were performed . C . The efficiency of infection ( 8 h ) of TgRHYFPSAG1 - OVA in Scramble and Rab22a KD JAWS - II DCs was measured by FACS analysis at the indicated MOI . Data represent mean ± SEM of duplicate values and are representative of three independent experiments . D . Immunoblotting showing the total amount of OVA in Scramble and Rab22a KD JAWS - II DCs after 90 min and 7 h of infection with TgRHYFPSAG1 - OVA parasite at MOI 2 and 6 . Fifty µg of total cell lysates were loaded onto each lane . Data are representative of two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Scramble and Rab22a KD JAWS-II DCs were infected with OVA-YFP-expressing T. gondii (TgRHYFPSAG1-OVA) for 8 h and confocal images detecting the parasite (green), endogenous Rab22a (red) and GRA6 (magenta) were taken. Top panels: Scramble cells; bottom panels: Rab22a KD cells. White boxes are shown at higher magnification in the insets. The nuclear marker DAPI (blue) and DIC images are shown in the left panels. Overlays are shown in the right panels. Scale bars: 5 µm. Images are representative of at least 30 analyzed from three independent experiments.B. The cross-presentation of OVA secreted by T. gondii (TgRHYFPSAG1-OVA) after 8 h of infection at the indicated MOI was evaluated by B3Z T cell activation. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. ***P < 0.001. A two-way ANOVA and the Bonferroni post-test were performed.C. The efficiency of infection (8 h) of TgRHYFPSAG1-OVA in Scramble and Rab22a KD JAWS-II DCs was measured by FACS analysis at the indicated MOI. Data represent mean ± SEM of duplicate values and are representative of three independent experiments.D. Immunoblotting showing the total amount of OVA in Scramble and Rab22a KD JAWS-II DCs after 90 min and 7 h of infection with TgRHYFPSAG1-OVA parasite at MOI 2 and 6. Fifty µg of total cell lysates were loaded onto each lane. Data are representative of two independent experiments.


Figure 1A ) Purified His - α - synuclein protein was agitated for 0 - 2 weeks ( lane 1 , 0weeks ; lanes 2 and 3 , 1week ; lanes 4 and 5 , 2weeks ) . Aliquots from each sample were separated into supernatant ( lanes 2 and 4 ) and pellet ( lanes3 and 5 ) fractions . These fractions were subjected to immunoblotting with anti - α - synuclein antibody . Numbers at left are molecular weight markers ( MW ) in kilodaltons . ( B ) Fibril formation of α - synuclein was measured with ProteoStat , an aggregation detection dye . a . u . , arbitrary units . ( C ) AFM images of α - synuclein agitated for 0 or 2 weeks were acquired in air using the dynamic mode . A high - magnification view of the boxed area is shown in the lower panel . The scale on the right represents the height of pixels in the image . Scale bars , 100 nm . ( D ) α - Synuclein inclusions in fibril - introduced HEK293 cells were detected immunocytochemically with anti - phosphorylated α - synuclein ( P - αSyn ) , anti - ubiquitin ( Ub ) , and anti - p62 ( p62 ) antibodies . Confocal images reconstructed in the z - axis along the white lines are shown in the right panel ( Z - axis ) . Blue , DAPI . Scale bar , 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) Purified His-α-synuclein protein was agitated for 0-2 weeks (lane 1, 0weeks; lanes 2 and 3, 1week; lanes 4 and 5, 2weeks). Aliquots from each sample were separated into supernatant (lanes 2 and 4) and pellet (lanes3 and 5) fractions. These fractions were subjected to immunoblotting with anti-α-synuclein antibody. Numbers at left are molecular weight markers (MW) in kilodaltons.(B) Fibril formation of α-synuclein was measured with ProteoStat, an aggregation detection dye. a.u., arbitrary units.(C) AFM images of α-synuclein agitated for 0 or 2 weeks were acquired in air using the dynamic mode. A high-magnification view of the boxed area is shown in the lower panel. The scale on the right represents the height of pixels in the image. Scale bars, 100 nm.(D) α-Synuclein inclusions in fibril-introduced HEK293 cells were detected immunocytochemically with anti-phosphorylated α-synuclein (P-αSyn), anti-ubiquitin (Ub), and anti-p62 (p62) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm.


Figure 2 ( A ) Autophagic clearance of α - synuclein inclusions was verified by immunocytochemical analysis with anti - phosphorylated α - synuclein ( P - αSyn ) and anti - LC3 ( LC3 ) antibodies . Confocal images reconstructed in the z - axis along the white lines are shown in the right panel ( Z - axis ) . Blue , DAPI . Scale bar , 10 µm . Phosphorylated α - synuclein - positive inclusions were sequestrated into LC3 - positive autophagosomes . ( B ) Similarly , this sequestration was confirmed in HEK293 cells stably expressing DsRed - LC3 . The upper and lower panels show α - synuclein fibrils - and mock - introduced cells , respectively . Blue , DAPI . Scale bar , 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Autophagic clearance of α-synuclein inclusions was verified by immunocytochemical analysis with anti-phosphorylated α-synuclein (P-αSyn) and anti-LC3 (LC3) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm. Phosphorylated α-synuclein-positive inclusions were sequestrated into LC3-positive autophagosomes.(B) Similarly, this sequestration was confirmed in HEK293 cells stably expressing DsRed-LC3. The upper and lower panels show α-synuclein fibrils-and mock-introduced cells, respectively. Blue, DAPI. Scale bar, 10 µm.


Figure 3 ( A ) HEK293 cells stably expressing GFP - LC3 were transfected with control ( upper ) or Atg - 5 siRNA ( lower ) . After 36 h , α - synuclein fibrils were introduced into them for 4 h , followed by immunostaining with anti - phospho - α - synuclein antibody ( P - αSyn ) . Blue , DAPI . Scale bar , 10 µm . ( B ) The number of GFP - LC3 - positive α - synuclein inclusions ( % ) were assessed in randomly chosen fields ( n = 5 ) with 48 - 141 inclusions . Statistical analysis was performed with t - test ( * p < 0 . 01 ) . ( C ) Clearance of α - synuclein inclusions in control ( solid bar ) or Atg - 5 ( open bar ) knockdown cells was measured at 4 h and 24 h after α - synuclein fibrils . The number of phosphorylated α - synuclein - positive cells was assessed in randomly chosen fields ( n = 5 ) . Data from each experiment are were normalized to Cont - siRNA - 4 h ( 100 % ) and represented as relative value of α - synuclein inclusions ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) HEK293 cells stably expressing GFP-LC3 were transfected with control (upper) or Atg-5 siRNA (lower). After 36 h, α-synuclein fibrils were introduced into them for 4 h, followed by immunostaining with anti-phospho-α-synuclein antibody (P-αSyn). Blue, DAPI. Scale bar, 10 µm.(B) The number of GFP-LC3-positive α-synuclein inclusions (%) were assessed in randomly chosen fields (n = 5) with 48-141 inclusions. Statistical analysis was performed with t-test (*p<0.01).(C) Clearance of α-synuclein inclusions in control (solid bar) or Atg-5 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment are were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01


Figure 4 ( A ) Autophagy flux was analyzed by LC3 protein monitoring . α - Synuclein fibrils - introduced HEK293 cells were treated with ( + ) / without ( − ) bafilomycin A1 ( BafA1 ) for 2 h . Cell lysates were subjected to immunoblotting analysis using anti - LC3 ( upper panel ) or anti - actin ( lower panel ) antibodies . Quantification of the relative levels of LC3 - II / Actin or autophagic flux ( Baf + / Baf - ) is shown with the ratio . Relative level of LC3 - II / Actin was represented by mean ± s . d . as a graph . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 05 . This experiment was repeated three times . ( B ) α - Synuclein fibrils were introduced into HEK293 cells , followed by addition of DMSO , 100 nM bafilomycin ( BafA1 ) , or 200 nM rapamycin ( Rapa ) . Cells were stained by anti - phosphorylated α - synuclein antibody ( green ) and DAPI ( blue ) 1 h ( left panels ) or 24 h ( right panels ) after introduction . ( C ) The number of phosphorylated α - synuclein - positive cells per unit area ( 0 . 6 mm2 ) was assessed in randomly chosen fields ( n = 6 ) . Data represent mean ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 05 , * * p < 0 . 001 . ( D ) Colocalization of α - synuclein inclusions and lysosomes . α - Synuclein inclusions ( P - αSyn ) and lysosomes ( LAMP1 ) were immunostained with anti - phospho - α - synuclein polyclonal antibody ( green ) and anti - LAMP1 antibody ( red ) 1 h ( left panel ) or 4 h ( middle panel ) after fibril introduction . Fibril - unintroduced cells were used as mock control ( right panel ) . Arrowheads and arrows indicate localized or unlocalized α - synuclein inclusions to lysosomes , respectively . Blue , DAPI . Scale bars , 10 µm . ( E ) Colocalization of α - synuclein inclusions ( n = 41 - 73 ) to lysosomes were measured in randomly chosen fields . The experiment was repeated three times . Data represent mean ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 01 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Autophagy flux was analyzed by LC3 protein monitoring. α-Synuclein fibrils-introduced HEK293 cells were treated with (+)/without (−) bafilomycin A1 (BafA1) for 2 h. Cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin or autophagic flux (Baf+/Baf-) is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.05. This experiment was repeated three times.(B) α-Synuclein fibrils were introduced into HEK293 cells, followed by addition of DMSO, 100 nM bafilomycin (BafA1), or 200 nM rapamycin (Rapa). Cells were stained by anti-phosphorylated α-synuclein antibody (green) and DAPI (blue) 1 h (left panels) or 24 h (right panels) after introduction.(C) The number of phosphorylated α-synuclein-positive cells per unit area (0.6 mm2) was assessed in randomly chosen fields (n = 6). Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.05, **p<0.001.(D) Colocalization of α-synuclein inclusions and lysosomes. α-Synuclein inclusions (P-αSyn) and lysosomes (LAMP1) were immunostained with anti-phospho-α-synuclein polyclonal antibody (green) and anti-LAMP1 antibody (red) 1 h (left panel) or 4 h (middle panel) after fibril introduction. Fibril-unintroduced cells were used as mock control (right panel). Arrowheads and arrows indicate localized or unlocalized α-synuclein inclusions to lysosomes, respectively. Blue, DAPI. Scale bars, 10 µm.(E) Colocalization of α-synuclein inclusions (n = 41-73) to lysosomes were measured in randomly chosen fields. The experiment was repeated three times. Data represent mean ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01.


Figure 5 ( A ) HEK293 cells stably expressing GFP - LC3 were transfected with p62 - siRNA ( right panels ) or control - siRNA ( left panels ) . Phosphorylated α - synuclein - positive inclusions ( P - αSyn ) are indicated by arrowheads . In p62 - knockdown cells , the expression and accumulation of p62 were not detected , and inclusions were never sequestrated by GFP - LC3 autophagosomes . Scale bars , 20 µm . ( B ) The number of GFP - LC3 - positive α - synuclein inclusions ( % ) in Fig . 5A were assessed in randomly chosen fields with 32 - 44 inclusions ( n = 3 ) . Data represent mean ± s . e . m . Statistical analysis was performed with t - test ( * p < 0 . 05 ) . ( C ) α - Synuclein fibrils were introduced into control ( Cont ) or p62 - knockdown ( p62 ) cells . Four hours after introduction , cell lysates were subjected to immunoblotting analysis using anti - LC3 ( upper panel ) or anti - actin ( lower panel ) antibodies . Quantification of the relative levels of LC3 - II / Actin is shown with the ratio . Relative level of LC3 - II / Actin was represented by mean ± s . d . as a graph . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 01 . This experiment was repeated three times . ( D ) Clearance of α - synuclein inclusions in control ( solid bar ) or p62 ( open bar ) knockdown cells was measured at 4 h and 24 h after α - synuclein fibrils . The number of phosphorylated α - synuclein - positive cells was assessed in randomly chosen fields ( n = 5 ) . Data from each experiment were normalized to Cont - siRNA - 4 h ( 100 % ) and represented as relative value of α - synuclein inclusions ± s . e . m . Statistical analysis was performed with one - way ANOVA ( post - hoc Tukey ' s test ) . * p < 0 . 01 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) HEK293 cells stably expressing GFP-LC3 were transfected with p62-siRNA (right panels) or control-siRNA (left panels). Phosphorylated α-synuclein-positive inclusions (P-αSyn) are indicated by arrowheads. In p62-knockdown cells, the expression and accumulation of p62 were not detected, and inclusions were never sequestrated by GFP-LC3 autophagosomes. Scale bars, 20 µm.(B) The number of GFP-LC3-positive α-synuclein inclusions (%) in Fig. 5A were assessed in randomly chosen fields with 32-44 inclusions (n = 3). Data represent mean ± s.e.m. Statistical analysis was performed with t-test (*p<0.05).(C) α-Synuclein fibrils were introduced into control (Cont) or p62-knockdown (p62) cells. Four hours after introduction, cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01. This experiment was repeated three times.(D) Clearance of α-synuclein inclusions in control (solid bar) or p62 (open bar) knockdown cells was measured at 4 h and 24 h after α-synuclein fibrils. The number of phosphorylated α-synuclein-positive cells was assessed in randomly chosen fields (n = 5). Data from each experiment were normalized to Cont-siRNA-4 h (100%) and represented as relative value of α-synuclein inclusions ± s.e.m. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.01.


Figure 6 ( A ) α - Synuclein fibrils were introduced into HEK293 cells stably expressing both DsRed - LC3 and EGFP - parkin . After 24 h , the cells were immunostained with anti - phosphorylated α - synuclein ( P - αSyn ) . There was no alteration in the localization of EGFP - parkin . The arrows indicate α - synuclein inclusion . Scale bar , 10 µm . ( B ) Mitochondrial clearance was confirmed in mock - ( upper panels ) or α - synuclein fibrils - introduced cells ( lower panels ) . After introduction , EGFP - parkin cells were treated with DMSO ( left ) or 10 µM CCCP for 4 h ( middle ) or 16 h ( right ) . Cells were examined immunocytochemically with anti - Tom20 ( red ) and anti - phosphorylated α - synuclein antibody ( P - αSyn ; aqua ) . Figures were presented as merged images . Individual images were provided in supplemental figure ( Fig . S8 ) . Scale bar , 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) α-Synuclein fibrils were introduced into HEK293 cells stably expressing both DsRed-LC3 and EGFP-parkin. After 24 h, the cells were immunostained with anti-phosphorylated α-synuclein (P-αSyn). There was no alteration in the localization of EGFP-parkin. The arrows indicate α-synuclein inclusion. Scale bar, 10 µm.(B) Mitochondrial clearance was confirmed in mock- (upper panels) or α-synuclein fibrils-introduced cells (lower panels). After introduction, EGFP-parkin cells were treated with DMSO (left) or 10 µM CCCP for 4 h (middle) or 16 h (right). Cells were examined immunocytochemically with anti-Tom20 (red) and anti-phosphorylated α-synuclein antibody (P-αSyn; aqua). Figures were presented as merged images. Individual images were provided in supplemental figure (Fig. S8). Scale bar, 10 µm.


Figure 3 ( Left panel ) A10 cells were transfected with Flag - FMRP for 3 hr then cultured for 24 hr prior to fixation and staining for β - catenin ( green ) , Flag ( FMRP , red ) , and nucleus ( blue ) . ( Bottom right panel ) A line was drawn through a representative cell to indicate relative intensity of RGB signals . ( Top right panel ) An orthogonal projection was generated by a Z - stack ( 240 nm interval ) image set using the ZEN program ( Zeiss ) depicting the region surrounding the nucleus in the XY , XZ , and YZ planesB . ( i ) HEK 293T cells were transfected with Flag - FMRP as in A , and endogenous β - catenin protein complexes were immunoprecipitated with β - catenin antibody . Co - immunoprecipitated Flag - FMRP protein was detected by Western blot analysis with the Flag antibody . Non - programmed rabbit IgG was used as a control . ( ii ) Primary VSMCs were harvested and subjected to an endogenous coIP analysis with either β - catenin or FMRP antibody . The corresponding protein was detected in the precipitated immunocomplex by Western blot . Non - programmed rabbit IgG was used as a control ( i ) Purified 6x - His - FMRP and various regions of β - catenin proteins fused to GST were incubated with glutathione - agarose beads . Purified GST or GST - fusion proteins were visualized by Western blotting using the GST antibody , and sizes of corresponding GST - fusion proteins were confirmed . Pulled - down FMRP proteins by GST fused β - catenin were detected by immunoblotting with the FMRP antibody . ( ii ) Interaction between FMRP and β - catenin was assessed in the presence or absence of RNA . HEK 293T cells were subjected to an endogenous coIP analysis using either β - catenin or FMRP antibody , in the presence or absence of RNase A ( 10 µg / mL ) . The corresponding protein was detected in the precipitated immunocomplex by Western blot , and non - programmed rabbit IgG was used as a control . RNA was extracted from parallel cultured cells and total RNA content was analyzed by agarose gel electrophoresis in the presence or absence of RNase A ( Top panel ) A10 cells were transfected with GBP - Lifeact and either EYFP - β - catenin , Flag - FMRP , or mCherry - β - catenin . The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag ( red ) , as indicated . ( Bottom panel ) A10 cells were transfected with GBP - Lifeact similar to above , but with both EYFP - β - catenin and Flag - FMRP , and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag ( red ) ( Top panel ) A10 cells were transfected wit EYFP - β - catenin , Flag - FMRP , or mCherry - β - catenin . The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag ( red ) , as indicated . ( Bottom panel ) A10 cells were transfected with GBP - Lifeact similar to above , but with both EYFP - β - catenin and Flag - FMRP , and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag ( red ) E . A10 cells were transfected as in ( D ) with GBP - Lamin

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(Left panel) A10 cells were transfected with Flag-FMRP for 3 hr then cultured for 24 hr prior to fixation and staining for β-catenin (green), Flag (FMRP, red), and nucleus (blue). (Bottom right panel) A line was drawn through a representative cell to indicate relative intensity of RGB signals. (Top right panel) An orthogonal projection was generated by a Z-stack (240 nm interval) image set using the ZEN program (Zeiss) depicting the region surrounding the nucleus in the XY, XZ, and YZ planesB. (i) HEK 293T cells were transfected with Flag-FMRP as in A, and endogenous β-catenin protein complexes were immunoprecipitated with β-catenin antibody. Co-immunoprecipitated Flag-FMRP protein was detected by Western blot analysis with the Flag antibody. Non-programmed rabbit IgG was used as a control. (ii) Primary VSMCs were harvested and subjected to an endogenous coIP analysis with either β-catenin or FMRP antibody. The corresponding protein was detected in the precipitated immunocomplex by Western blot. Non-programmed rabbit IgG was used as a control(i) Purified 6x-His-FMRP and various regions of β-catenin proteins fused to GST were incubated with glutathione-agarose beads. Purified GST or GST-fusion proteins were visualized by Western blotting using the GST antibody, and sizes of corresponding GST-fusion proteins were confirmed. Pulled-down FMRP proteins by GST fused β-catenin were detected by immunoblotting with the FMRP antibody. (ii) Interaction between FMRP and β-catenin was assessed in the presence or absence of RNA. HEK 293T cells were subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody, in the presence or absence of RNase A (10 µg/mL). The corresponding protein was detected in the precipitated immunocomplex by Western blot, and non-programmed rabbit IgG was used as a control. RNA was extracted from parallel cultured cells and total RNA content was analyzed by agarose gel electrophoresis in the presence or absence of RNase A(Top panel) A10 cells were transfected with GBP-Lifeact and either EYFP-β-catenin, Flag-FMRP, or mCherry-β-catenin. The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag (red), as indicated. (Bottom panel) A10 cells were transfected with GBP-Lifeact similar to above, but with both EYFP-β-catenin and Flag-FMRP, and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag (red)(Top panel) A10 cells were transfected wit EYFP-β-catenin, Flag-FMRP, or mCherry-β-catenin. The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag (red), as indicated. (Bottom panel) A10 cells were transfected with GBP-Lifeact similar to above, but with both EYFP-β-catenin and Flag-FMRP, and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag (red) E. A10 cells were transfected as in (D) with GBP-Lamin B1


Figure 4A . Co - sedimentation of protein / RNA complexes on a 10 - 50 % sucrose gradient . Cell lysates generated from the cells transfected with either siRNA targeting fmr1 or its scrambled control were ultracentrifuged in sucrose gradients ; peaks corresponding to the 40S and 60S subunits , 80S monosome , and polysomes were detected by UV absorbance at 254 nm , and indicated proteins in these fractions were detected by Western blot . Corresponding total cell lysate was used as input ( i ) Precipitated proteins in A10 cell lysates with m7GTP - agarose beads were identified by Western blot analysis . eIF4E and tubulin were shown as positive and negative controls , respectively . Total lysates were used as input . ( ii ) A10 cell lysates were incubated with GTP - agarose beads , and none of the proteins tested were precipitated with the beads . Total lysates were used as input control ( i ) HEK 293T cells were transfected with empty vector or Flag - FMRP , and lysates were subjected to m7GTP - agarose pull - down as in ( C ) ( ii ) HEK 293T cells were transfected with empty vector or Flag - FMRP , and lysates were subjected to GTP - agarose pull - down as in ( i ) . E . HEK 293T cells were transfected with either siRNAs targeting fmr1 or scrambled control and were subjected to m7GTP - agarose pull - downs as in ( C ) F . HEK 293T cells lysates were subjected to m7GTP - agarose pull - downs as in ( C ) in the presence or absence of RNase A ( 10 µg / mL ) . RNA was extracted from parallel lysates and RNA content was analyzed by agarose gel electrophoresis .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Co-sedimentation of protein/RNA complexes on a 10-50% sucrose gradient. Cell lysates generated from the cells transfected with either siRNA targeting fmr1 or its scrambled control were ultracentrifuged in sucrose gradients; peaks corresponding to the 40S and 60S subunits, 80S monosome, and polysomes were detected by UV absorbance at 254 nm, and indicated proteins in these fractions were detected by Western blot. Corresponding total cell lysate was used as input(i) Precipitated proteins in A10 cell lysates with m7GTP-agarose beads were identified by Western blot analysis. eIF4E and tubulin were shown as positive and negative controls, respectively. Total lysates were used as input. (ii) A10 cell lysates were incubated with GTP-agarose beads, and none of the proteins tested were precipitated with the beads. Total lysates were used as input control(i) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to m7GTP-agarose pull-down as in (C)(ii) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to GTP-agarose pull-down as in (i). E. HEK 293T cells were transfected with either siRNAs targeting fmr1 or scrambled control and were subjected to m7GTP-agarose pull-downs as in (C)F. HEK 293T cells lysates were subjected to m7GTP-agarose pull-downs as in (C) in the presence or absence of RNase A (10 µg/mL). RNA was extracted from parallel lysates and RNA content was analyzed by agarose gel electrophoresis.


Figure 5Primary VSMCs ( left panel ) , A10 ( middle panel ) , and HEK 293T cells ( right panel ) were transfected with siRNAs targeting ctnnb1 ( β - catenin ) or scrambled RNA . After 36 hr , the cells were pulsed with 0 . 5 µM puromycin for 15 min and then harvested . The puromycin incorporated peptides were detected by Western blot analysis with puromycin antibody . β - catenin blots indicated efficacy of siRNA and actin was used as a loading control . The average puromycin / actin ratio ( n = 3 ) is shown in the graphs to the right of each cell type and the error bars indicate standard deviation . A one - way ANOVA and Tukey post - hoc test was performed , with p - values indicated above the error barsA10 cells were transfected with Flag - FMRP then treated with either cycloheximide or its solvent ( DMSO ) for 4 hr prior to fixation . β - catenin ( green ) , Flag ( FMRP , red ) were visualized by immunofluorescence . Nucleus ( blue ) was identified by DNA staining with Hoechst 33342 . Arrows indicate a loss of β - catenin signal from the perinuclear region that was seen in the control following cycloheximide treatmentA10 cells were transfected with Flag - FMRP as above and then pre - treated with rapamycin or solvent ( DMSO ) for 1 hr in serum - free media . Serum was added for 2 hr before the cells were harvested and subjected to coIP analysis using β - catenin antibody . Non - programmed rabbit IgG was used as a control . Existence of Flag - FMRP in the immunocomplex was detected by Flag antibody and β - catenin by GFP antibodyA10 cells were transfected with Flag - FMRP followed by rapamycin treatment similar to ( D ) . Cells were fixed and stained for β - catenin ( green ) , Flag ( FMRP , red ) , and nucleus ( blue ) . A loss of β - catenin signal from the perinuclear region that was seen in the control is indicated by the arrows .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Primary VSMCs (left panel), A10 (middle panel), and HEK 293T cells (right panel) were transfected with siRNAs targeting ctnnb1 (β-catenin) or scrambled RNA. After 36 hr, the cells were pulsed with 0.5 µM puromycin for 15 min and then harvested. The puromycin incorporated peptides were detected by Western blot analysis with puromycin antibody. β-catenin blots indicated efficacy of siRNA and actin was used as a loading control. The average puromycin/actin ratio (n=3) is shown in the graphs to the right of each cell type and the error bars indicate standard deviation. A one-way ANOVA and Tukey post-hoc test was performed, with p-values indicated above the error barsA10 cells were transfected with Flag-FMRP then treated with either cycloheximide or its solvent (DMSO) for 4 hr prior to fixation. β-catenin (green), Flag (FMRP, red) were visualized by immunofluorescence. Nucleus (blue) was identified by DNA staining with Hoechst 33342. Arrows indicate a loss of β-catenin signal from the perinuclear region that was seen in the control following cycloheximide treatmentA10 cells were transfected with Flag-FMRP as above and then pre-treated with rapamycin or solvent (DMSO) for 1 hr in serum-free media. Serum was added for 2 hr before the cells were harvested and subjected to coIP analysis using β-catenin antibody. Non-programmed rabbit IgG was used as a control. Existence of Flag-FMRP in the immunocomplex was detected by Flag antibody and β-catenin by GFP antibodyA10 cells were transfected with Flag-FMRP followed by rapamycin treatment similar to (D). Cells were fixed and stained for β-catenin (green), Flag (FMRP, red), and nucleus (blue). A loss of β-catenin signal from the perinuclear region that was seen in the control is indicated by the arrows.


Figure 6 ( Top panel ) Primary VSMCs were serum deprived overnight prior to stimulation with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . The cells were treated with 0 . 5 µM puromycin for 15 min and harvested for Western blot analysis . Total β - catenin levels are indicated , and actin was used as a loading control . The average puromycin / actin ratio ( n = 3 ) was graphed . Error bars indicate standard deviation . An independent samples t - test was performed ( 2 - tailed ) , with the p - value indicated above the error bar . ( Bottom panel ) Cells were treated similarly to above and fixed and stained for β - catenin ( green ) and nucleus ( blue ) The experiment in ( A ) was repeated using A10 cells ( i ) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . The cells were harvested and subjected to an endogenous coIP analysis using either β - catenin or FMRP antibody . The corresponding proteins in the precipitated immunocomplex were detected by Western blot analysis . Non - programed rabbit IgG was used as a control . Efficacy of Wnt - 3a treatment was assessed by inhibition of GSK3 mediated phosphorylation of β - catenin ( S33 / 37 / T41 ) by Wnt - 3a . ( ii ) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . Cell lysates were then subjected to m7GTP - agarose pull - down analysis . eIF4E and tubulin were used as positive and negative controls , respectively , and phospho - β - catenin ( S33 / 37 / T41 ) Western blots were used as controls for Wnt - 3a stimulation .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(Top panel) Primary VSMCs were serum deprived overnight prior to stimulation with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were treated with 0.5 µM puromycin for 15 min and harvested for Western blot analysis. Total β-catenin levels are indicated, and actin was used as a loading control. The average puromycin/actin ratio (n=3) was graphed. Error bars indicate standard deviation. An independent samples t-test was performed (2-tailed), with the p-value indicated above the error bar. (Bottom panel) Cells were treated similarly to above and fixed and stained for β-catenin (green) and nucleus (blue) The experiment in (A) was repeated using A10 cells(i) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were harvested and subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody. The corresponding proteins in the precipitated immunocomplex were detected by Western blot analysis. Non-programed rabbit IgG was used as a control. Efficacy of Wnt-3a treatment was assessed by inhibition of GSK3 mediated phosphorylation of β-catenin (S33/37/T41) by Wnt-3a. (ii) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. Cell lysates were then subjected to m7GTP-agarose pull-down analysis. eIF4E and tubulin were used as positive and negative controls, respectively, and phospho-β-catenin (S33/37/T41) Western blots were used as controls for Wnt-3a stimulation.


Figure 1 A mRNA expression in FACS - isolated Lin ( Ter119 / CD31 / CD45 ) - Sca1 + progenitor cells from female mouse subcutaneous fat , differentiated in the presence of 1 µM cPGI2 or vehicle for the indicated time , as determined by expression profiling ( n = 3 , E - MTAB - 3693 ) * * * * P = 3 * 10 - 6 ( Day 2 ) , * * * * P = 4 * 10 - 7 ( Day 4 ) , * * * * P = 1 * 10 - 6 ( Day 6 ) in 2x2 ANOVA with Bonferroni posttests ( cPGI2 vs . vehicle ) B mRNA expression in MACS - isolated Lin - Sca1 + progenitor cells from female mouse subcutaneous fat , differentiated in the presence of 100 nM Rosi or vehicle for the indicated time , as determined by qRT - PCR ( n = 4 ) * * * * P = 1 * 10 - 10 ( Day 1 and 2 ) , * * P = 0 . 001 , in 2x2 ANOVA with Bonferroni posttests ( Rosi vs . vehicle ) C mRNA expression in female Lin - Sca1 + cells , differentiated in the presence of 100 nM Rosi or vehicle for 8 days , as determined by qRT - PCR ( n = 3 ) t - test Cited4 - / - vs . Cited4 + / + ( Rosi ) * P = 0 . 013 ( Ucp1 ) , * * P = 0 . 004 ( Cpt1b ) , * P = 0 . 026 ( Dio2 ) D - F mRNA expression in primary SVF cells from human subcutaneous fat , differentiated in the presence of 100 nM Rosi ( D ) or vehicle ( D - F ) , as determined by qRT - PCR at the indicated time points ( n = 5 patients ) . ♀ / ♂ represent individual data ( D ) * * * * P = 3 * 10 - 5 ( Day 2 ) , * * * * P = 3 * 10 - 6 ( Day 6 ) , * * * * P = 4 * 10 - 9 ( Day 10 ) , * * * * P = 3 * 10 - 7 ( Day 14 ) , in 2x2 ANOVA with Bonferroni posttests ( Rosi vs . vehicle ) . ( E , F ) Pearson correlation coefficient ( r ) and P - value are shown G mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days , as determined by qRT - PCR ( n = 3 ) * * * P = 0 . 0002 ( CITED4 ) , * * P = 0 . 002 ( UCP1 ) , * P = 0 . 02 ( UCP1 ) , * P = 0 . 035 / 0 . 026 ( PPARG ) , * * * P = 0 . 0006 ( SLC2A4 ) , * * P = 0 . 002 ( ADIPOQ ) in one - way ANOVA with Tukey posttests ( vs . siCtrl )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 A mRNA expression in FACS-isolated Lin(Ter119/CD31/CD45)-Sca1+ progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 1 µM cPGI2 or vehicle for the indicated time, as determined by expression profiling (n=3, E-MTAB-3693) ****P=3*10-6 (Day 2), ****P=4*10-7 (Day 4), ****P=1*10-6 (Day 6) in 2x2 ANOVA with Bonferroni posttests (cPGI2 vs. vehicle) B mRNA expression in MACS-isolated Lin-Sca1+ progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT-PCR (n=4) ****P=1*10-10 (Day 1 and 2), **P=0.001, in 2x2 ANOVA with Bonferroni posttests (Rosi vs. vehicle) C mRNA expression in female Lin-Sca1+ cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT-PCR (n=3) t-test Cited4-/- vs. Cited4+/+ (Rosi) *P=0.013 (Ucp1), **P=0.004 (Cpt1b), *P=0.026 (Dio2) D-F mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi (D) or vehicle (D-F), as determined by qRT-PCR at the indicated time points (n=5 patients). ♀/♂ represent individual data (D) ****P=3*10-5 (Day 2), ****P=3*10-6 (Day 6), ****P=4*10-9 (Day 10), ****P=3*10-7 (Day 14), in 2x2 ANOVA with Bonferroni posttests (Rosi vs. vehicle). (E, F) Pearson correlation coefficient (r) and P-value are shown G mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT-PCR (n=3) ***P=0.0002 (CITED4), **P=0.002 (UCP1), *P=0.02 (UCP1), *P=0.035/0.026 (PPARG), ***P=0.0006 (SLC2A4), **P=0.002 (ADIPOQ) in one-way ANOVA with Tukey posttests (vs. siCtrl)


Figure 2 A - C Quantitative fluorescence microscopy of LipidTOX - and DAPI - stained female Cited4F / F Lin - Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days ( n = 5 ) . * * P = 0 . 002 in t - test ( Cre vs . Ctrl ) . Scale bar is 10 µm D , E Ucp1 expression in female Cited4F / F Lin - Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days , as determined by Western blot with VCP as loading control ( n = 3 ) F Cellular respiration in female Lin - Sca1 + progenitor cells differentiated in the presence of 100 nM Rosi for 8 days . The extracellular oxygen consumption rate ( OCR ) was determined upon injection of the indicated substances and normalized to DNA content . CL : CL - 316243

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 A-C Quantitative fluorescence microscopy of LipidTOX- and DAPI-stained female Cited4F/F Lin-Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days (n=5). **P=0.002 in t-test (Cre vs. Ctrl). Scale bar is 10 µm  D, E Ucp1 expression in female Cited4F/F Lin-Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days, as determined by Western blot with VCP as loading control (n=3)  F Cellular respiration in female Lin-Sca1+ progenitor cells differentiated in the presence of 100 nM Rosi for 8 days. The extracellular oxygen consumption rate (OCR) was determined upon injection of the indicated substances and normalized to DNA content. CL: CL-316243


Figure 4 A , B mRNA expression in scWAT of mice fed a diet with 0 . 0075 % Rosi or control diet for 2 . 5 weeks , determined by qRT - PCR t - test Cited4 - / - vs . Cited4 + / + ( Rosi ) , ( A ) n = 5 / 5 / 6 / 6 , * * P = 0 . 003 ( Ucp1 ) , * * P = 0 . 008 ( Cidea ) , * * P = 0 . 007 ( Cyc1 ) , * P = 0 . 048 ( Ndufb3 ) , ( B ) n = 5 / 4 / 5 / 5 , * P = 0 . 022 ( Ucp1 ) , * P = 0 . 037 ( Cidea ) C , D Ucp1 expression in scWAT of mice treated as in A , as determined by Western blot with VCP as loading control t - test Cited4 - / - vs . Cited4 + / + ( Rosi ) , females : n = 5 / 5 / 6 / 6 , * P = 0 . 011 , males : n = 5 / 4 / 5 / 5 E mRNA expression in scWAT of female mice treated with CL - 316 , 243 ( CL ) ( 1 mg / kg / day via Alzet minipumps ) or vehicle for 10 days ( n = 5 / 7 / 13 , t - test Cited4 - / - vs . Cited4 + / + ( CL ) ) F mRNA expression in scWAT of female mice exposed to 5 ° C or 23 ° C for 2 weeks ( n = 5 / 10 / 6 , t - test Cited4 - / - vs . Cited4 + / + ( 5 ° C ) )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 A, B mRNA expression in scWAT of mice fed a diet with 0.0075% Rosi or control diet for 2.5 weeks, determined by qRT-PCR t-test Cited4-/- vs. Cited4+/+ (Rosi), (A) n=5/5/6/6, **P=0.003 (Ucp1), **P=0.008 (Cidea), **P=0.007 (Cyc1), *P=0.048 (Ndufb3), (B) n=5/4/5/5, *P=0.022 (Ucp1), *P=0.037 (Cidea) C, D Ucp1 expression in scWAT of mice treated as in A, as determined by Western blot with VCP as loading control t-test Cited4-/- vs. Cited4+/+ (Rosi), females: n=5/5/6/6, *P=0.011, males: n=5/4/5/5 E mRNA expression in scWAT of female mice treated with CL-316,243 (CL) (1 mg/kg/day via Alzet minipumps) or vehicle for 10 days (n=5/7/13, t-test Cited4-/- vs. Cited4+/+ (CL))  F mRNA expression in scWAT of female mice exposed to 5°C or 23°C for 2 weeks (n=5/10/6, t-test Cited4-/- vs. Cited4+/+ (5°C))


Figure 5 A Oxygen consumption rate of female mice fed a diet with 0 . 0075 % Rosi for 2 . 5 weeks , determined by indirect calorimetry and normalized to body weight ( n = 9 / 10 ) . Grey bars represent the dark phase B Oxygen consumption rate of female mice shown in ( A ) adjusted for body weight by ANCOVA . 3 - day averages of VO2 were calculated for each mouse ( n = 9 / 10 ) . * P = 0 . 025 in ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + ) C Oxygen consumption rate of female mice fed a diet with 0 . 0075 % Rosi for 3 . 5 weeks and injected with 1 mg / kg CL - 316 , 243 ( CL ) at the indicated time point , as determined by indirect calorimetry ( n = 9 / 10 ) . * P = 0 . 013 ( - 1 h ) , * P = 0 . 045 ( 3 h ) , * P = 0 . 015 ( 5 h ) , * P = 0 . 016 ( 6 h ) , * P = 0 . 019 ( 8 h ) , * P = 0 . 046 ( 10 h ) in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) D Maximal CL - induced oxygen consumption rate of female mice shown in ( C ) adjusted for body weight by ANCOVA . Averages of VO2 throughout 3 - 8 hours post CL injection were calculated for each mouse ( n = 9 / 10 ) . * P = 0 . 049 in ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + ) E Oxygen consumption rate of male mice treated as in ( A ) , determined as in ( C ) ( n = 9 / 10 ) . ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + ) F Oxygen consumption rate of male mice treated as in ( C ) , determined as in ( C ) ( n = 9 / 10 ) . * P = 0 . 044 ( 9 h ) , * P = 0 . 013 ( 12 h ) in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) G Maximal CL - induced oxygen consumption rate of male mice shown in ( F ) and calculated as in ( D ) ( n = 9 / 10 ) . ANCOVA with Bonferroni posttest ( Cited4 - / - vs . Cited4 + / + )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 A Oxygen consumption rate of female mice fed a diet with 0.0075% Rosi for 2.5 weeks, determined by indirect calorimetry and normalized to body weight (n=9/10). Grey bars represent the dark phase  B Oxygen consumption rate of female mice shown in (A) adjusted for body weight by ANCOVA. 3-day averages of VO2 were calculated for each mouse (n=9/10). *P=0.025 in ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)  C Oxygen consumption rate of female mice fed a diet with 0.0075% Rosi for 3.5 weeks and injected with 1 mg/kg CL-316,243 (CL) at the indicated time point, as determined by indirect calorimetry (n=9/10). *P=0.013 (-1 h), *P=0.045 (3 h), *P=0.015 (5 h), *P=0.016 (6 h), *P=0.019 (8 h), *P=0.046 (10 h) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)  D Maximal CL-induced oxygen consumption rate of female mice shown in (C) adjusted for body weight by ANCOVA. Averages of VO2 throughout 3-8 hours post CL injection were calculated for each mouse (n=9/10). *P=0.049 in ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)  E Oxygen consumption rate of male mice treated as in (A), determined as in (C) (n=9/10). ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)  F Oxygen consumption rate of male mice treated as in (C), determined as in (C) (n=9/10). *P=0.044 (9 h), *P=0.013 (12 h) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)  G Maximal CL-induced oxygen consumption rate of male mice shown in (F) and calculated as in (D) (n=9/10). ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+)


Figure 6 A , B mRNA expression in scWAT of mice fed a high - fat diet ( HFD ) for 11 weeks followed by 5 weeks of HFD with 0 . 0075 % Rosi ( qRT - PCR ) . t - test Cited4 - / - vs . Cited4 + / + , ( A ) n = 8 / 7 , * P = 0 . 029 ( Ucp1 ) , * * P = 0 . 009 ( Elovl3 ) , * * P = 0 . 006 ( Cpt1b ) , * * P = 0 . 004 ( Cyc1 ) , * P = 0 . 016 ( Ndufb3 ) , * * P = 0 . 003 ( Cd36 ) , * * P = 0 . 009 ( Gyk ) , ( B ) n = 7 / 8 , * P = 0 . 026 ( Pck1 ) C , D Ucp1 protein expression in scWAT of mice treated as in ( A ) , determined by Western blot with VCP as loading control ( n = 8 / 7 for females and n = 7 / 8 for males ) . Arrow indicates the band specific to Ucp1 ( see Appendix Fig 4F )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 A, B mRNA expression in scWAT of mice fed a high-fat diet (HFD) for 11 weeks followed by 5 weeks of HFD with 0.0075% Rosi (qRT-PCR). t-test Cited4-/- vs. Cited4+/+, (A) n=8/7, *P=0.029 (Ucp1), **P=0.009 (Elovl3), **P=0.006 (Cpt1b), **P=0.004 (Cyc1), *P=0.016 (Ndufb3), **P=0.003 (Cd36), **P=0.009 (Gyk), (B) n=7/8, *P=0.026 (Pck1)  C, D Ucp1 protein expression in scWAT of mice treated as in (A), determined by Western blot with VCP as loading control (n=8/7 for females and n=7/8 for males). Arrow indicates the band specific to Ucp1 (see Appendix Fig 4F)


Figure 7 A Blood glucose during glucose tolerance test ( GTT ) on female mice after 11 weeks of HFD and 3 weeks of HFD + Rosi ( n = 8 ) . * * * P = 0 . 001 , in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) B Fasting serum insulin in female mice after 10 weeks of HFD ( " Before Rosi " ) or 11 weeks of HFD and 2 . 5 weeks of HFD + Rosi ( " After Rosi " ) ( n = 8 ) . * * * P = 0 . 0009 in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi ) ; P = 0 . 725 ( Cited4 - / - vs . Cited4 + / + Before Rosi ) C Blood glucose during GTT on male mice as in ( A ) ( n = 5 / 8 ) . Repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) D Fasting serum insulin in male mice as in ( C ) ( n = 7 / 7 / 8 / 8 ) . * * * P = 0 . 001 ( Cited4 + / + ) , * * * P = 0 . 0008 ( Cited4 - / - ) in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi ) E , F Blood glucose during insulin tolerance test ( ITT ) with 1 U insulin per kg body mass on female mice after 12 weeks of HFD ( n = 8 ) ( E ) or 16 weeks of HFD and 4 weeks of HFD + Rosi ( n = 7 ) ( F ) , expressed as % of the 0 - time point value . * * P = 0 . 003 in repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) . G Area under the curve ( AUC ) of blood glucose during the ITT ( 0 - 120 min ) in ( E , F ) . 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi ) H , I Blood glucose during insulin tolerance test ( ITT ) on male mice as in ( E , F ) , n = 8 ( H ) , n = 7 / 8 ( I ) . Repeated measures 2x2 ANOVA with Holm - Sidak posttests ( Cited4 - / - vs . Cited4 + / + ) . J Area under the curve ( AUC ) of blood glucose during the ITT in ( H , J ) . * * P = 0 . 001 , * * * P = 0 . 00006 in 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi ) K , L Fasting serum non - esterified fatty acids ( NEFA ) in female ( K ) or male ( L ) mice after 10 weeks of HFD ( " Before Rosi " ) or 11 weeks of HFD and 2 . 5 weeks of HFD + Rosi ( " After Rosi " ) , n = 7 / 8 / 8 / 8 ( K ) , n = 6 / 6 / 6 / 8 ( L ) . * * P = 0 . 001 in 2x2 ANOVA with Holm - Sidak posttests ( After Rosi vs . Before Rosi )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7 A Blood glucose during glucose tolerance test (GTT) on female mice after 11 weeks of HFD and 3 weeks of HFD+Rosi (n=8). ***P=0.001, in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)  B Fasting serum insulin in female mice after 10 weeks of HFD ("Before Rosi") or 11 weeks of HFD and 2.5 weeks of HFD+Rosi ("After Rosi") (n=8). ***P=0.0009 in repeated measures 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi); P=0.725 (Cited4-/- vs. Cited4+/+ Before Rosi)  C Blood glucose during GTT on male mice as in (A) (n=5/8). Repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+)  D Fasting serum insulin in male mice as in (C) (n=7/7/8/8). ***P=0.001 (Cited4+/+), ***P=0.0008 (Cited4-/-) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)  E, F Blood glucose during insulin tolerance test (ITT) with 1 U insulin per kg body mass on female mice after 12 weeks of HFD (n=8) (E) or 16 weeks of HFD and 4 weeks of HFD+Rosi (n=7) (F), expressed as % of the 0-time point value. **P=0.003 in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+). G Area under the curve (AUC) of blood glucose during the ITT (0-120 min) in (E,F). 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)  H, I Blood glucose during  insulin tolerance test (ITT) on male mice as in (E,F), n=8 (H), n=7/8 (I). Repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+). J Area under the curve (AUC) of blood glucose during the ITT in (H,J). **P=0.001, ***P=0.00006 in 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)  K, L Fasting serum non-esterified fatty acids (NEFA) in female (K) or male (L) mice after 10 weeks of HFD ("Before Rosi") or 11 weeks of HFD and 2.5 weeks of HFD+Rosi ("After Rosi"), n=7/8/8/8 (K), n=6/6/6/8 (L). **P=0.001 in 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi)


Figure 2 ( B ) Volcano plot showing differentially expressed genes between WT ( WT1 and WT2 ) and patient ( V . 1 ( F1 ) , V . 5 ( F1 ) ) primary dermal fibroblasts . The vertical axis ( y - axis ) shows the - log10 P - value , whereas the horizontal axis ( x - axis ) displays the log2 fold change value . The red dots represent the upregulated transcripts ; the blue dots represent the downregulated transcripts . A total of 172 genes were found significantly dysregulated . TAPT1 , a gene located in the IBD region , appeared among the most significantly downregulated genes in the patients .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Volcano plot showing differentially expressed genes between WT (WT1 and WT2) and patient (V.1 (F1), V.5 (F1)) primary dermal fibroblasts. The vertical axis (y-axis) shows the -log10 P-value, whereas the horizontal axis (x-axis) displays the log2 fold change value. The red dots represent the upregulated transcripts; the blue dots represent the downregulated transcripts. A total of 172 genes were found significantly dysregulated. TAPT1, a gene located in the IBD region, appeared among the most significantly downregulated genes in the patients.


Figure 4 ( B ) qPCR results using specific primers for TAPT1 and TAPT1 - AS1 in 3 WT ( WT1 , WT2 , V . 2 ( F1 ) ) and 3 affected ( V . 1 ( F1 ) , V . 5 ( F1 ) , IV . 1 ( F2 ) ) primary fibroblasts . TAPT1 mRNA is significantly reduced in all patients compared to WTs , whereas TAPT1 - AS1 transcript levels are unaffected . Fold change relative to V . 2 ( F1 ) is plotted as mean ± SD . Asterisks indicate conventional statistical significance ( Student t - test ; n . s . p - value > 0 . 05 , * * * * p - value < 0 . 0001 ) . ( C ) Western blot analysis of endogenous TAPT1 protein ( ~ 60 kDa ) using whole protein extracts from primary dermal fibroblasts from WT ( WT1 and WT2 ) , heterozygous ( IV . 3 ( F1 ) ) and homozygous ( V . 1 ( F1 ) , V . 5 ( F1 ) and IV . 1 ( F2 ) ) individuals and two different commercial antibodies ( top : Sigma , HPA042567 ; bottom : Sigma , HPA048658 ) . Results show a complete absence of TAPT1 protein in patient samples . GAPDH was used as a loading control . ( D ) qPCR analysis of TAPT1 expression in 3 WT ( WT1 , WT2 , WT3 ) and 3 affected ( V . 1 ( F1 ) , V . 5 ( F1 ) , IV . 1 ( F2 ) ) primary fibroblasts treated with cycloheximide ( CHX ) . CHX was used to block nonsense mediated decay ( NMD ) . Our results showed a time dependent increase in the level of TAPT1 transcripts in all 3 patient cells while TAPT1 RNA level remained constant in the WT cells . For each graph , fold change relative to non - treated condition is plotted as mean ± SD . Asterisks indicate conventional statistical significance ( Student t - test ; n . s . p - value > 0 . 05 , * * * * p - value < 0 . 0001 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) qPCR results using specific primers for TAPT1 and TAPT1-AS1 in 3 WT (WT1, WT2 , V.2 (F1)) and 3 affected (V.1 (F1), V.5 (F1), IV.1 (F2)) primary fibroblasts. TAPT1 mRNA is significantly reduced in all patients compared to WTs, whereas TAPT1-AS1 transcript levels are unaffected. Fold change relative to V.2 (F1) is plotted as mean ± SD. Asterisks indicate conventional statistical significance (Student t-test; n.s. p-value > 0.05, **** p-value < 0.0001).(C) Western blot analysis of endogenous TAPT1 protein (~60 kDa) using whole protein extracts from primary dermal fibroblasts from WT (WT1 and WT2), heterozygous (IV.3 (F1)) and homozygous (V.1 (F1), V.5 (F1) and IV.1 (F2)) individuals and two different commercial antibodies (top: Sigma, HPA042567; bottom: Sigma, HPA048658). Results show a complete absence of TAPT1 protein in patient samples. GAPDH was used as a loading control.(D) qPCR analysis of TAPT1 expression in 3 WT (WT1, WT2, WT3) and 3 affected (V.1 (F1), V.5 (F1), IV.1 (F2)) primary fibroblasts treated with cycloheximide (CHX). CHX was used to block nonsense mediated decay (NMD). Our results showed a time dependent increase in the level of TAPT1 transcripts in all 3 patient cells while TAPT1 RNA level remained constant in the WT cells. For each graph, fold change relative to non-treated condition is plotted as mean ± SD. Asterisks indicate conventional statistical significance (Student t-test; n.s. p-value > 0.05, **** p-value < 0.0001).


Figure 5 ( B ) Western blot analyses for TAPT1 ( ~ 60 kDa ) using cytosolic , Mito / ER / Golgi and nuclear protein extracts from primary dermal fibroblasts of two WTs ( WT1 and WT2 ) and two patients ( V . 5 ( F1 ) and IV . 1 ( F2 ) ) . TAPT1 protein is highly enriched in the Mito / ER / Golgi fraction , and to a lower extent in the nuclear fraction . GAPDH , TGN46 and BiP served as a cytosolic , Golgi network and ER markers , respectively . Adenylate Kinase ( AK2 ) was used as a mitochondrial marker . Laminin A / C was used as a nuclear marker . ( C ) Immunofluorescence staining of mitochondria using anti - TOM20 ( green ) , ER using anti - CANX ( red ) and Golgi using anti - GLG1 ( red ) in primary dermal fibroblasts from WT1 and V . 5 ( F1 ) . Similar staining patterns are observed with the three antibodies in both cell lines . Scale bar represents 10 µm . ( D ) qPCR analysis of a panel of canonical ER stress markers shows no significant differences in 3 patients ( - / - ) ( V . I ( F1 ) , V . 5 ( F1 ) and IV . 1 ( F2 ) ) primary dermal fibroblasts compared to WTs ( + / + ) ( WT1 , WT2 and WT3 ) cells . Fold change relative to WT is plotted as mean ± SD . Statistical significance was tested by Student t - test ( n . s . p - value > 0 . 05 ) . ( E ) CMV cell infection assay on 2 patient ( V . 1 ( F1 ) and V . 5 ( F1 ) ) and 3 WT ( WT1 , WT4 and WT5 ) primary dermal fibroblast cell lines , using β - galactosidase activity as a readout . MRC5 cell line was used as a positive control . All of the cells were infected by the HCMV strain RC256 at a MOI = 0 . 1 . Data are shown as mean ± SD of three technical replicates . Statistical significance was tested by Student t - test ( n . s . p - value > 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Western blot analyses for TAPT1 (~60 kDa) using cytosolic, Mito/ER/Golgi and nuclear protein extracts from primary dermal fibroblasts of two WTs (WT1 and WT2) and two patients (V.5 (F1) and IV.1 (F2)). TAPT1 protein is highly enriched in the Mito/ER/Golgi fraction, and to a lower extent in the nuclear fraction. GAPDH, TGN46 and BiP served as a cytosolic, Golgi network and ER markers, respectively. Adenylate Kinase (AK2) was used as a mitochondrial marker. Laminin A/C was used as a nuclear marker.(C) Immunofluorescence staining of mitochondria using anti-TOM20 (green), ER using anti-CANX (red) and Golgi using anti-GLG1 (red) in primary dermal fibroblasts from WT1 and V.5 (F1). Similar staining patterns are observed with the three antibodies in both cell lines. Scale bar represents 10 µm.(D) qPCR analysis of a panel of canonical ER stress markers shows no significant differences in 3 patients (-/-) (V.I (F1), V.5 (F1) and IV.1 (F2)) primary dermal fibroblasts compared to WTs (+/+) (WT1, WT2 and WT3) cells. Fold change relative to WT is plotted as mean ± SD. Statistical significance was tested by Student t-test (n.s. p-value > 0.05).(E) CMV cell infection assay on 2 patient (V.1 (F1) and V.5 (F1)) and 3 WT (WT1, WT4 and WT5) primary dermal fibroblast cell lines, using β-galactosidase activity as a readout. MRC5 cell line was used as a positive control. All of the cells were infected by the HCMV strain RC256 at a MOI=0.1. Data are shown as mean ± SD of three technical replicates. Statistical significance was tested by Student t-test (n.s. p-value > 0.05).


Figure 6 ( A ) Volcano plot showing differentially expressed genes as determined by RNA - seq in patient primary dermal fibroblasts ( V . 1 ( F1 ) , V . 5 ( F1 ) ) compared to WT ( WT1 and WT2 ) cells . The y - axis shows the - log10 P - value , whereas the x - axis displays the log2 fold change value . The red dots represent 75 significantly upregulated genes , and the blue dots represent 97 significantly downregulated genes . ( B ) qPCR validation test for 3 top dysregulated genes ( RARRES2 , ZIC1 , and ZIC4 ) detected by RNA - seq . The analysis was performed on RNA samples independent from those sent for RNA - seq for 2 WTs ( WT1 and WT2 ) and 2 patients ( V . 5 ( F1 ) and IV . 1 ( F2 ) ) . Fold change relative to WT1 is plotted as mean ± SD of three technical replicates . Asterisks indicate statistical significance ( Student t - test ; * * * p - value < 0 . 001 , * * * * p - value < 0 . 0001 ) . ( C ) Volcano plot showing genes with an altered occupancy of transcriptionally engaged Pol II in patients ( V . 1 ( F1 ) , V . 5 ( F1 ) ) compared to WT ( WT1 and WT2 ) primary fibroblast cells using SI - NET - seq . The y - axis shows the - Log10 P - value , whereas the x - axis indicates the log2 fold change value for the Pol II occupancy . The Pol II density is increased in 222 genes ( red dots ) , and decreased in 95 genes ( blue dots ) . The yellow dot represents TAPT1 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Volcano plot showing differentially expressed genes as determined by RNA-seq in patient primary dermal fibroblasts (V.1 (F1), V.5 (F1)) compared to WT (WT1 and WT2) cells. The y-axis shows the -log10 P-value, whereas the x-axis displays the log2 fold change value. The red dots represent 75 significantly upregulated genes, and the blue dots represent 97 significantly downregulated genes.(B) qPCR validation test for 3 top dysregulated genes (RARRES2, ZIC1, and ZIC4) detected by RNA-seq. The analysis was performed on RNA samples independent from those sent for RNA-seq for 2 WTs (WT1 and WT2) and 2 patients (V.5 (F1) and IV.1 (F2)). Fold change relative to WT1 is plotted as mean ± SD of three technical replicates. Asterisks indicate statistical significance (Student t-test; *** p-value < 0.001, **** p-value < 0.0001).(C) Volcano plot showing genes with an altered occupancy of transcriptionally engaged Pol II in patients (V.1 (F1), V.5 (F1)) compared to WT (WT1 and WT2) primary fibroblast cells using SI-NET-seq. The y-axis shows the -Log10 P-value, whereas the x-axis indicates the log2 fold change value for the Pol II occupancy. The Pol II density is increased in 222 genes (red dots), and decreased in 95 genes (blue dots). The yellow dot represents TAPT1.


Figure 1The VPR and DPR ( right ) were dissected from E10 . 5 Pdx1 - GFP embryos ( left ) . Scale bar : 500 µm . DPR , dorsal Pdx1 - expressing region ; VPR , ventral Pdx1 - expressing region ; Duo , duodenum bud . FACS gating for sorting GFPlow and GFPhigh cells from the VPR and DPR . The ratios of GFPhigh cells to GFP + cells in the VPR and DPR . The data show the mean ± SEM ; n = number of independent biological replicates , unpaired t - test . * * * p - value < 0 . 001 . Hierarchical clustering of 815 genes correlating with the first two PCs identified three groups of cell types and four major distinct clusters ( a - d ) of genes ( p < 1 × 10 - 9 , cell cycle - related genes are excluded ) . Each column is a single - cell , and each row represents one gene . Cluster - specific TFs are listed on the right . The genes in bold are known to have roles in pancreatic development . L , GFPlow ; H , GFPhigh .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1The VPR and DPR (right) were dissected from E10.5 Pdx1-GFP embryos (left). Scale bar: 500 µm. DPR, dorsal Pdx1-expressing region; VPR, ventral Pdx1-expressing region; Duo, duodenum bud.FACS gating for sorting GFPlow and GFPhigh cells from the VPR and DPR.The ratios of GFPhigh cells to GFP+ cells in the VPR and DPR. The data show the mean ± SEM; n = number of independent biological replicates, unpaired t-test. ***p-value < 0.001.Hierarchical clustering of 815 genes correlating with the first two PCs identified three groups of cell types and four major distinct clusters (a-d) of genes (p < 1 ×10-9, cell cycle-related genes are excluded). Each column is a single-cell, and each row represents one gene. Cluster-specific TFs are listed on the right. The genes in bold are known to have roles in pancreatic development. L, GFPlow; H, GFPhigh.