Figure 9 ( A ) Images showing UHMK1 , p - NCOA3 ( S1062 / T1067 ) , and ATIC protein expression in two human GC specimens . Scale bars = 100μm . ( B ) Pearson correlation analysis about of the levels of UHMK1 , p - NCOA3 ( S1062 / T1067 ) and ATIC proteins in patients with GC ( n = 120 ) by IHC . ( C ) ( Ci ) OS data from GC patients stratified by the level of UHMK1 . ( Cii ) Combination of UHMK1 and p - NCOA3 ( S1062 / T1067 ) expression predicts more exact OS of GC patients . Survival curves were plotted according to Kaplan - Meier method , with the log - rank test applied for comparison .

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 O O O O O O O

Figure 9(A) Images showing UHMK1, p-NCOA3 (S1062/T1067), and ATIC protein expression in two human GC specimens. Scale bars=100μm. (B) Pearson correlation analysis about of the levels of UHMK1, p-NCOA3 (S1062/T1067) and ATIC proteins in patients with GC (n=120) by IHC. (C) (Ci)OS data from GC patients stratified by the level of UHMK1. (Cii)Combination of UHMK1 and p-NCOA3 (S1062/T1067) expression predicts more exact OS of GC patients. Survival curves were plotted according to Kaplan-Meier method, with the log-rank test applied for comparison.


Figure 1D Representative confocal images of HEK293 cells expressing various GFP - fused proteins ( green ) and supplemented with TopFluor ® TMR - PS ( red ) ; scale bar = 10 µm . Yellow arrows indicate high intensity PS fluorescent regions . E % PM with PS clusters = area of high intensity fluorescent PS clusters over total plasma membrane area from images in panel D . Black bars are control proteins and blue bars are eVP40 proteins . Values are reported as mean ± s . d . ; N > 18 cells per biological replicate , n = 3 for 3 biological replicates ; A one - way ANOVA was performed with multiple comparisons compared to the control GFP % PS clustering ( * * * p = 0 . 0007 , * * p = 0 . 004 ) . Data information : PS : phosphatidylserine ; PM : plasma membrane .

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

Figure 1D Representative confocal images of HEK293 cells expressing various GFP-fused proteins (green) and supplemented with TopFluor® TMR-PS (red); scale bar = 10 µm. Yellow arrows indicate high intensity PS fluorescent regions. E %PM with PS clusters = area of high intensity fluorescent PS clusters over total plasma membrane area from images in panel D. Black bars are control proteins and blue bars are eVP40 proteins. Values are reported as mean ± s.d.; N>18 cells per biological replicate, n=3 for 3 biological replicates; A one-way ANOVA was performed with multiple comparisons compared to the control GFP %PS clustering (***p=0.0007, **p=0.004). Data information: PS: phosphatidylserine; PM: plasma membrane.


Figure 2D , E PS concentration in LUVs enhances the ability of His6 - eVP40 to oligomerize on membranes . D Representative western blot of chemical crosslinking performed on His6 - WT - eVP40 following incubation with LUVs of varying PS content ( detected by Mouse α - His antibody & HRP - Sheep α - Mouse ) . E Oligomerization capacity was determined from the western blot band density ratio of oligomers / ( monomer + dimer ) from chemical crosslinking experiments . A one - way ANOVA was performed with multiple comparisons compared to the control 0 % PS LUVs control ( 30 % PS * p = 0 . 02 ; 60 % PS * p = 0 . 017 ) . n = 3 biological replicates . Values are reported as mean ± s . d . ; Data information : LUVs : large unilamellar vesicles ; PS : phosphatidylserine ; HRP : horseradish peroxidase .

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

Figure 2D, E PS concentration in LUVs enhances the ability of His6-eVP40 to oligomerize on membranes. D Representative western blot of chemical crosslinking performed on His6-WT-eVP40 following incubation with LUVs of varying PS content (detected by Mouse α-His antibody & HRP-Sheep α-Mouse). E Oligomerization capacity was determined from the western blot band density ratio of oligomers/(monomer + dimer) from chemical crosslinking experiments. A one-way ANOVA was performed with multiple comparisons compared to the control 0% PS LUVs control (30% PS *p= 0.02; 60% PS *p=0.017). n=3 biological replicates. Values are reported as mean ± s.d.; Data information: LUVs: large unilamellar vesicles; PS: phosphatidylserine; HRP: horseradish peroxidase.


Figure 3D , E Analysis of PS clustering in HEK293 cells in response to fendiline treatment through N & B analysis . D Left panel : Representative images from time - lapse ( 30 frames ) imaging of HEK293 expressing GFP - LactC2 and treated with fendiline for 48 hours ; scale bar = 5 µm . Middle panel : Brightness and Intensity plots for each representative image . Right panel : Selection map correlating each pixel in the representative image to an oligomerization state ( b value ) ( red : monomer - 5mer , green : 5mer - 10mer , blue : > 10mer ) . E Average % pixels quantification from panel ( d ) = Percentage of GFP - LactC2 with brightness values corresponding to monomer - 5mer ( ~ 1 . - 1 . 5 ) , 5mer - 10mer ( ~ 1 . 5 - 1 . 9 ) and > 10mer ( > 1 . 9 ) over the total pixels within each image . Values are reported as mean ± s . d . ; N ≥ 9 , n = 3 biological replicates ; A two - way ANOVA was performed with Dunnett ' s multiple comparisons comparted to the control DMSO % average pixels ( * * * * p < 0 . 0001 , * * p = 0 . 0043 ) . Data information : GFP - LactC2 : phosphatidylserine sensor ; N & B : Number & Brightness analysis ; PS : phosphatidylserine .

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 O O O O O O O O O O O O O O O O O O O O O O O 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 3D, E Analysis of PS clustering in HEK293 cells in response to fendiline treatment through N&B analysis. D Left panel: Representative images from time-lapse (30 frames) imaging of HEK293 expressing GFP-LactC2 and treated with fendiline for 48 hours; scale bar= 5 µm. Middle panel: Brightness and Intensity plots for each representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization state (b value) (red: monomer-5mer, green: 5mer-10mer, blue: >10mer). E Average % pixels quantification from panel (d)= Percentage of GFP-LactC2 with brightness values corresponding to monomer-5mer (~1.-1.5), 5mer-10mer (~1.5-1.9) and >10mer (>1.9) over the total pixels within each image. Values are reported as mean ± s.d.; N≥9, n=3 biological replicates; A two-way ANOVA was performed with Dunnett's multiple comparisons comparted to the control DMSO % average pixels (****p<0.0001, **p=0.0043). Data information: GFP-LactC2: phosphatidylserine sensor; N&B: Number & Brightness analysis; PS: phosphatidylserine.


Figure 4Effect of fendiline on EBOV infection . A Representative confocal images of Vero E6 cells infected with EBOV ( Kikwit ) at the indicated MOI and treated with the indicated concentration of fendiline . Cells were pretreated 24 hours prior to infection with the indicated concentration of fendiline . Post infection , cells were treated 1 hour later ( d - 1 / 0 ) , treated every day ( e . d ) , or treated every other day ( e . o . d ) and fixed at either 48 hours , 72 hours or 96 hours post infection . ( green = EBOV ; blue = nuclei ) . White numbering in top right corner indicates % infection . Scale bar = 100 μm . Data information : EBOV : Ebola virus ; MOI : multiplicity of infectionEffect of fendiline on EBOV infection . B - D Quantification of % inhibition of EBOV by fendiline . B 48 hours ( MOI 1 . 0 ) C 72 hours ( MOI 0 . 1 ) D 96 hours ( MOI 0 . 1 ) . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons was performed . n = 3 biological replicates . Data information : EBOV : Ebola virus ; MOI : multiplicity of infection ; d . - 1 / 0 : treatment 1 hour after infection ; e . d . : treatment every day ; e . o . d . : treatment every other day .

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 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

Figure 4Effect of fendiline on EBOV infection. A Representative confocal images of Vero E6 cells infected with EBOV (Kikwit) at the indicated MOI and treated with the indicated concentration of fendiline. Cells were pretreated 24 hours prior to infection with the indicated concentration of fendiline. Post infection, cells were treated 1 hour later (d -1/0), treated every day (e.d), or treated every other day (e.o.d) and fixed at either 48 hours, 72 hours or 96 hours post infection. (green=EBOV; blue= nuclei). White numbering in top right corner indicates %infection. Scale bar = 100 μm. Data information: EBOV: Ebola virus; MOI: multiplicity of infectionEffect of fendiline on EBOV infection. B-D Quantification of % inhibition of EBOV by fendiline. B 48 hours (MOI 1.0) C 72 hours (MOI 0.1) D 96 hours (MOI 0.1). Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons was performed. n=3 biological replicates. Data information: EBOV: Ebola virus; MOI: multiplicity of infection; d. -1/0: treatment 1 hour after infection; e.d.: treatment every day; e.o.d.: treatment every other day.


Figure 5A - C Effect of fendiline on eVP40 and mVP40 PM localization in HEK293 cells after 48 hours of treatment . A Representative confocal images from live cell imaging experiments of HEK293 cells expressing EGFP - WT - eVP40 ( top panel ) and EGFP - WT - mVP40 ( bottom panel ) after 48 hours of fendiline treatment . scale bars = 10 µm . Effect of fendiline on mVP40 ( B ) and eVP40 ( C ) PM localization was quantified by the ratio of EGFP fluorescence intensity at the PM / total EGFP fluorescence intensity ( and normalized to DMSO control ) . N > 15 cell per biological replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA with multiple comparisons was performed compared to the DMSO control . Data information : PM : plasma membraneD , E Analysis of eVP40 oligomerization in HEK293 cells in response to 48 hour fendiline treatment using N & B analysis . D Left panel : Representative images from time - lapse ( 30 frames ) of HEK293 expressing EGFP - WT - eVP40 and treated with fendiline for 48 hours . scale bar = 5 µm . Middle panel : Brightness and Intensity plots for each representative image . Right panel : Selection map correlating each pixel in the representative image to an oligomerization state ( b value ) ( red : monomer - hexamer , green : hexamer - 12mer , blue : 12mer - 24mer , pink : > 24mer ) . E Average % pixel quantification from panel ( d ) = % of GFP - WT - eVP40 with brightness values corresponding to monomer - hexamer ( ~ 1 . - 1 . 6 ) , hexamer - 12mer ( ~ 1 . 6 - 2 . 0 ) , 12mer - 24mer ( 2 . 0 - 3 . 2 ) and > 24mer ( > 3 . 2 ) over the total pixels within each image . Values are reported as mean ± s . d . ; N ≥ 9 cell per biological replicate , n = 3 biological replicates ; A two - way ANOVA was performed with Dunnett ' s multiple comparisons compared to the control DMSO % average pixels ( * * p = 0 . 0035 ) . Data information : N & B : Number & Brightness analysis .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5A-C Effect of fendiline on eVP40 and mVP40 PM localization in HEK293 cells after 48 hours of treatment. A Representative confocal images from live cell imaging experiments of HEK293 cells expressing EGFP-WT-eVP40 (top panel) and EGFP-WT-mVP40 (bottom panel) after 48 hours of fendiline treatment. scale bars= 10 µm. Effect of fendiline on mVP40 (B) and eVP40 (C) PM localization was quantified by the ratio of EGFP fluorescence intensity at the PM / total EGFP fluorescence intensity (and normalized to DMSO control). N>15 cell per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA with multiple comparisons was performed compared to the DMSO control. Data information: PM: plasma membraneD, E Analysis of eVP40 oligomerization in HEK293 cells in response to 48 hour fendiline treatment using N&B analysis. D Left panel: Representative images from time-lapse (30 frames) of HEK293 expressing EGFP-WT-eVP40 and treated with fendiline for 48 hours. scale bar = 5 µm. Middle panel: Brightness and Intensity plots for each representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization state (b value) (red: monomer-hexamer, green: hexamer-12mer, blue: 12mer-24mer, pink: >24mer). E Average % pixel quantification from panel (d)= % of GFP-WT-eVP40 with brightness values corresponding to monomer-hexamer (~1.-1.6), hexamer-12mer (~1.6-2.0), 12mer-24mer (2.0-3.2) and >24mer (>3.2) over the total pixels within each image. Values are reported as mean ± s.d.; N≥9 cell per biological replicate, n=3 biological replicates; A two-way ANOVA was performed with Dunnett's multiple comparisons compared to the control DMSO % average pixels (**p=0.0035). Data information: N&B: Number & Brightness analysis.


Figure 6A - D Functional budding assays assessed at 24 hours ( A - B ) and 48 hours ( C - D ) post treatment . A Representative western blot of budding assays performed at 24 hours post transfection with vehicle control ( DMSO ) treatment or 500 nM , 1 μM , or 5 μM fendiline treatment . VLP samples ( top panel ) and cell lysate samples ( bottom panel ) collected from HEK293 cells and immunoblotted for eVP40 expression ; GAPDH served as a loading control . eVP40 detected by ( Rabbit α - eVP40 and HRP - Goat α - Rabbit ) ; GAPDH detected by mouse α - GAPDH and HRP - Sheep α - Mouse ) B Quantification of relative budding index at 24 hours post vehicle or fendiline treatment . Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction / ( total eVP40 cell lysate + eVP40 VLP band density ) and was normalized to the DMSO control . Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification . n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons compared to the DMSO control . C Representative western blot of budding assays performed at 48 hours post vehicle control ( DMSO ) treatment or 500 nM , 1 μM , or 5 μM fendiline treatment . VLP samples ( top panel ) and cell lysate samples ( bottom panel ) collected from HEK293 cells and immunoblotted for eVP40 expression ; GAPDH served as a loading control . eVP40 detected by ( Rabbit α - eVP40 and HRP - Goat α - Rabbit ) ; GAPDH detected by ( Mouse α - GAPDH and HRP - Sheep α - Mouse ) D Quantification of relative budding index at 48 hours post fendiline treatment . Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction / ( total eVP40 cell lysate + eVP40 VLP band density ) and was normalized to the DMSO control . Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification . n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons compared to the DMSO control . ( * p = 0 . 0260 ) Data information : VLPs : virus - like particles ; GAPDH : glyceraldehyde 3 - phosphate dehydrogenase ; HRP : horseradish peroxidase .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6A-D Functional budding assays assessed at 24 hours (A-B) and 48 hours (C-D) post treatment. A Representative western blot of budding assays performed at 24 hours post transfection with vehicle control (DMSO) treatment or 500 nM, 1 μM, or 5 μM fendiline treatment. VLP samples (top panel) and cell lysate samples (bottom panel) collected from HEK293 cells and immunoblotted for eVP40 expression; GAPDH served as a loading control. eVP40 detected by (Rabbit α-eVP40 and HRP-Goat α-Rabbit); GAPDH detected by mouse α-GAPDH and HRP-Sheep α-Mouse) B Quantification of relative budding index at 24 hours post vehicle or fendiline treatment. Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction/(total eVP40 cell lysate + eVP40 VLP band density) and was normalized to the DMSO control. Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification. n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO control. C Representative western blot of budding assays performed at 48 hours post vehicle control (DMSO) treatment or 500 nM, 1 μM, or 5 μM fendiline treatment. VLP samples (top panel) and cell lysate samples (bottom panel) collected from HEK293 cells and immunoblotted for eVP40 expression; GAPDH served as a loading control. eVP40 detected by (Rabbit α-eVP40 and HRP-Goat α-Rabbit); GAPDH detected by (Mouse α-GAPDH and HRP-Sheep α-Mouse) D Quantification of relative budding index at 48 hours post fendiline treatment. Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction/(total eVP40 cell lysate + eVP40 VLP band density) and was normalized to the DMSO control. Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification. n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO control. (*p=0.0260) Data information: VLPs: virus-like particles; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HRP: horseradish peroxidase.


Figure 7A - C TEM analysis of eVLP morphology . A Representative transmission electron micrographs of eVLPs purified from HEK293 cells expressing FLAG - eVP40 and eGP following 48 hours of DMSO ( left panel ) or 5 µM fendiline treatment ( right panel ) . B Quantification of eVLP length ( µm ) of DMSO - derived eVLPs ( black ) and fendiline - derived eVLPs ( blue ) . N > 50 VLPs per biological replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A two - tailed t - test was performed ( * * p = 0 . 0139 ) . C Quantification of eVLP diameter ( nm ) of DMSO - derived eVLPs ( black ) and fendiline - derived eVLPs ( blue ) . N > 50 VLPs per biological replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A two - tailed t - test was performed ( * p = 0 . 0430 ) . Data information : TEM : transmission electron microscopy ; eVLPs : entry - competent VLPs ; eGP : Ebola virus glycoproteinD , E Fluorescence based DiI TIM - 1 dependent entry assay . D Representative confocal images from the DiI - entry assay comparing entry of eVLPs produced from DMSO ( top panel ) and fendiline - treated HEK293 cells ( bottom panel ) into target cells ( HEK293 cells transiently expressing increasing amounts of TIM - 1 ; 0 . 0 µg , 0 . 5 µg , 1 . 0 µg ) . A stack of 10 frames was acquired for each image . DiI ( initially red ) was recolored to yellow for easier observation in print ; blue ( Hoechst 3342 stain ) ; scale bar = 10 µm . E Quantification of eVLP entry was performed by calculating the total number of DiI punctate / the total number of DiI - positive cells . Three images from each z - stack were quantified . N = 9 cell per replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons against the 0 . 0 µg TIM - 1 condition for both DMSO - and fendiline derived eVLPs . ( * * * * p < 0 . 0001 ; * * p = 0 . 0093 ) . Data information : TIM - 1 : t - cell immunoglobulin receptor - 1 ; eVLPs : entry - competent VLPs ; DiI : 1 , 1 ' - Dioctadecyl - 3 , 3 , 3 ' , 3 ' - Tetramethylindocarbocyanine Perchlorate .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7A-C TEM analysis of eVLP morphology. A Representative transmission electron micrographs of eVLPs purified from HEK293 cells expressing FLAG-eVP40 and eGP following 48 hours of DMSO (left panel) or 5 µM fendiline treatment (right panel). B Quantification of eVLP length (µm) of DMSO-derived eVLPs (black) and fendiline-derived eVLPs (blue). N>50 VLPs per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A two-tailed t-test was performed (**p=0.0139). C Quantification of eVLP diameter (nm) of DMSO-derived eVLPs (black) and fendiline-derived eVLPs (blue). N>50 VLPs per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A two-tailed t-test was performed (*p=0.0430). Data information: TEM: transmission electron microscopy; eVLPs: entry-competent VLPs; eGP: Ebola virus glycoproteinD, E Fluorescence based DiI TIM-1 dependent entry assay. D Representative confocal images from the DiI-entry assay comparing entry of eVLPs produced from DMSO (top panel) and fendiline-treated HEK293 cells (bottom panel) into target cells (HEK293 cells transiently expressing increasing amounts of TIM-1; 0.0 µg, 0.5 µg, 1.0 µg). A stack of 10 frames was acquired for each image. DiI (initially red) was recolored to yellow for easier observation in print; blue (Hoechst 3342 stain); scale bar = 10 µm. E Quantification of eVLP entry was performed by calculating the total number of DiI punctate / the total number of DiI-positive cells. Three images from each z-stack were quantified. N=9 cell per replicate, n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons against the 0.0 µg TIM-1 condition for both DMSO- and fendiline derived eVLPs.(****p<0.0001; **p=0.0093). Data information: TIM-1: t-cell immunoglobulin receptor-1; eVLPs: entry-competent VLPs; DiI: 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate.


Figure 1B . Before differentiation , hESCs show uniformly high expression of OCT4 . Treatment with BMP4 produces a mixture of OCT4 + / CDX2 - self - renewing hESCs and OCT4 - / CDX2 + mesodermal cells . C . Quantification of OCT4 and CDX2 expression by immunofluorescence after 24 h of BMP4 treatment reveals two populations of hESCs . Cells were assigned to one of two distinct populations based on a 2 - component mixed Gaussian distributionE . Filmstrip of OCT4 dynamics in an undifferentiated hESC throughout its cell cycle duration . Yellow outlines indicate the region used to quantify mean nuclear fluorescence intensity . F . Distribution of OCT4 levels in individual hESCs . A single OCT4 level was quantified for each cell by averaging the mean nuclear mCherry intensity over the lifetime of the cell . G . Single - cell traces of OCT4 signaling . The length of each cell ' s trace indicates its cell cycle duration . H . Distribution of cell cycle durations for 120 hESCs .

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

Figure 1B. Before differentiation, hESCs show uniformly high expression of OCT4. Treatment with BMP4 produces a mixture of OCT4+/CDX2- self-renewing hESCs and OCT4-/CDX2+ mesodermal cells.C. Quantification of OCT4 and CDX2 expression by immunofluorescence after 24 h of BMP4 treatment reveals two populations of hESCs. Cells were assigned to one of two distinct populations based on a 2-component mixed Gaussian distributionE. Filmstrip of OCT4 dynamics in an undifferentiated hESC throughout its cell cycle duration. Yellow outlines indicate the region used to quantify mean nuclear fluorescence intensity.F. Distribution of OCT4 levels in individual hESCs. A single OCT4 level was quantified for each cell by averaging the mean nuclear mCherry intensity over the lifetime of the cell.G. Single-cell traces of OCT4 signaling. The length of each cell's trace indicates its cell cycle duration.H. Distribution of cell cycle durations for 120 hESCs.


Figure 2A . Single - cell traces of hESCs before and after treatment with 100 ng / mL BMP4 . Cells were imaged for 42 h prior to BMP4 treatment . Mean nuclear OCT4 levels were quantified every 5 minutes and individual cells were tracked from the cell division event that created the cell until its own division . B . 24 h after BMP4 treatment , cells were fixed , stained for expression of CDX2 , and returned to the microscope for registration with the final time - lapse image . Mean nuclear OCT4 - mCherry and CDX2 were quantified for each cell , and the resulting distribution was fit to a 2 - component mixed Gaussian distribution representing self - renewing ( OCT4 + / CDX2 - ) and differentiated ( OCT4 - / CDX2 + ) cells hESCs that could be assigned to either distribution with > 99 % confidence ( red and green dots , Pdiff < 0 . 01 or Pdiff > 0 . 99 ) were considered for pro - fate analysis . Cells that did not reach this threshold ( gray dots ) were not used to determine pro - fate . Because cells in the final frame were assigned to only self - renewing or differentiated categories , pro - mixed cellsC . Distributions of OCT4 levels in pro - self - renewing , pro - mixed , and pro - differentiated cell populations . D . Distributions of OCT4 pulse frequencies in pro - pluripotent , pro - mixed , and pro - differentiated cell populations . To gain an unbiased look at preexisting determinants of cell fate in panels only cells who completed their entire cell cycle duration before BMP4 addition ( t = 0 ) were included in the analysis . E . Distributions of cell cycle durations in pro - pluripotent , pro - mixed , and pro - differentiated cell populations . To gain an unbiased look at preexisting determinants of cell fate in panels , only cells who completed their entire cell cycle duration before BMP4 addition ( t = 0 ) were included in the analysis . F . Decision surfaces for logistic regression classifier . Cells with self - renewing ( red ) or differentiated ( green ) pro - fates are plotted above the predicted class surfaces . ( left ) Slice of decision surface for pulse frequency vs . OCT4 levels at a cell cycle duration of 14 h . ( right ) Slice of decision surface for cell cycle duration vs . OCT4 level at a pulse frequency of 0 . 25 h - 1 . * P < 0 . 05 , * * P < 0 . 0005 ; ns , not significant .

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 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 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 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 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. Single-cell traces of hESCs before and after treatment with 100 ng/mL BMP4. Cells were imaged for 42 h prior to BMP4 treatment. Mean nuclear OCT4 levels were quantified every 5 minutes and individual cells were tracked from the cell division event that created the cell until its own division.B. 24 h after BMP4 treatment, cells were fixed, stained for expression of CDX2, and returned to the microscope for registration with the final time-lapse image. Mean nuclear OCT4-mCherry and CDX2 were quantified for each cell, and the resulting distribution was fit to a 2-component mixed Gaussian distribution representing self-renewing (OCT4+/CDX2-) and differentiated (OCT4-/CDX2+) cells hESCs that could be assigned to either distribution with >99% confidence (red and green dots, Pdiff < 0.01 or Pdiff > 0.99) were considered for pro-fate analysis. Cells that did not reach this threshold (gray dots) were not used to determine pro-fate. Because cells in the final frame were assigned to only self-renewing or differentiated categories, pro-mixed cellsC. Distributions of OCT4 levels in pro-self-renewing, pro-mixed, and pro-differentiated cell populations.D. Distributions of OCT4 pulse frequencies in pro-pluripotent, pro-mixed, and pro-differentiated cell populations. To gain an unbiased look at preexisting determinants of cell fate in panels only cells who completed their entire cell cycle duration before BMP4 addition (t = 0) were included in the analysis.E. Distributions of cell cycle durations in pro-pluripotent, pro-mixed, and pro-differentiated cell populations. To gain an unbiased look at preexisting determinants of cell fate in panels , only cells who completed their entire cell cycle duration before BMP4 addition (t = 0) were included in the analysis.F. Decision surfaces for logistic regression classifier. Cells with self-renewing (red) or differentiated (green) pro-fates are plotted above the predicted class surfaces. (left) Slice of decision surface for pulse frequency vs. OCT4 levels at a cell cycle duration of 14 h. (right) Slice of decision surface for cell cycle duration vs. OCT4 level at a pulse frequency of 0.25 h-1. * P < 0.05, ** P < 0.0005; ns, not significant.


Figure 3A . Lineage of OCT4 expression dynamics . Mean nuclear OCT4 levels were quantified in individual hESCs continuously for 72 h under undifferentiated conditions . Vertical bars represent individual cells that completed an entire cell cycle lifetime . Thin horizontal bars denote cell division events . Color scale indicates low ( black ) , intermediate ( red ) , and high ( white ) OCT4 expression levels . B . Differences in OCT4 levels between sister cells , cousin cells , second cousin cells , and randomly paired cells . C . Filmstrip showing distribution of OCT4 to daughter cells during cell division . D . Distribution of OCT4 ratios between sister cells before and after BMP4 treatment . Ratios for both sister cells ( r and 1 / r ) are plotted to emphasize central tendency toward r = 1 : 1 . Differently colored curves represent the distribution of ratios at different time points after division . For time points after 5 minutes , the ratio was determined by first calculating a mean OCT4 level for each sister cell among all previous time points and then calculating the resulting ratio between sisters . E . Proliferating H9 - OCT4 - mCherry hESCs were imaged for 24 h , fixed , and stained for DNA content with DAPI . The probability density of DAPI ratio ( blue ) and OCT4 - mCherry ratio ( red ) between sister cells that divided ≤ 1 h prior to fixation was calculated for 151 cell pairs using a normal kernel function . A two - sample Kolmogorov - Smirnov test was used to determine significance . * P < 0 . 05 , * * * P < 0 . 0005 , two - sample Kolmogorov - Smirnov test ; ns , not significant .

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 B-PANEL_START 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 O O O O O O O O O O O O O O O O O O O O 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 3A. Lineage of OCT4 expression dynamics. Mean nuclear OCT4 levels were quantified in individual hESCs continuously for 72 h under undifferentiated conditions. Vertical bars represent individual cells that completed an entire cell cycle lifetime. Thin horizontal bars denote cell division events. Color scale indicates low (black), intermediate (red), and high (white) OCT4 expression levels.B. Differences in OCT4 levels between sister cells, cousin cells, second cousin cells, and randomly paired cells.C. Filmstrip showing distribution of OCT4 to daughter cells during cell division.D. Distribution of OCT4 ratios between sister cells before and after BMP4 treatment. Ratios for both sister cells (r and 1/r) are plotted to emphasize central tendency toward r = 1:1. Differently colored curves represent the distribution of ratios at different time points after division. For time points after 5 minutes, the ratio was determined by first calculating a mean OCT4 level for each sister cell among all previous time points and then calculating the resulting ratio between sisters.E. Proliferating H9-OCT4-mCherry hESCs were imaged for 24 h, fixed, and stained for DNA content with DAPI. The probability density of DAPI ratio (blue) and OCT4-mCherry ratio (red) between sister cells that divided ≤1 h prior to fixation was calculated for 151 cell pairs using a normal kernel function. A two-sample Kolmogorov-Smirnov test was used to determine significance. * P < 0.05, *** P < 0.0005, two-sample Kolmogorov-Smirnov test; ns, not significant.


Figure 4B . OCT4 mixing as a function of cell history . 30 randomly chosen cells were colored according to their OCT4 expression level at 8 h after division . C . Variance in final OCT4 expression explained over time during maternal , inherited , and autonomous periods of cell history . At each time point , the coefficient of determination ( R2 ) between total OCT4 expression levels and final OCT4 expression at 8 h was calculated . D . Estimate of variance explained by asymmetric distribution of OCT4 to daughter cells . The same R2 over time mother - daughter traces were randomly sampled from a set of mother - daughter traces that contained sister - cell pairs ( blue line ) or only one randomly selected sister cell from each pair ( red line ) . E . Difference in variance explained between red and blue traces in panel D . The black line represents the actual difference ; the red line shows a smoothing ( moving average = 1 . 25 h ) of the black trace to highlight the overall trend .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4B. OCT4 mixing as a function of cell history. 30 randomly chosen cells were colored according to their OCT4 expression level at 8 h after division.C. Variance in final OCT4 expression explained over time during maternal, inherited, and autonomous periods of cell history. At each time point, the coefficient of determination (R2) between total OCT4 expression levels and final OCT4 expression at 8 h was calculated.D. Estimate of variance explained by asymmetric distribution of OCT4 to daughter cells. The same R2 over time mother-daughter traces were randomly sampled from a set of mother-daughter traces that contained sister-cell pairs (blue line) or only one randomly selected sister cell from each pair (red line). E. Difference in variance explained between red and blue traces in panel D. The black line represents the actual difference; the red line shows a smoothing (moving average = 1.25 h) of the black trace to highlight the overall trend.


Figure 1Meth Induces Rapid Alkalization of Acidic Organelles , and Changes in Endosomal Structure in Dendritic Cells ( A ) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment . Similar results were obtained in three independent experiments . ( Statistics n = 25 - 35 , mean ± SEM , * * p < 0 . 001 , * * * p < 0 . 0001 , two - tailed ANOVA . ) ( B ) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence ( ) or presence ( ▴ ) of 50 μM Meth . p < 0 . 001 for all time points versus control ( n = 25 - 35 , mean ± SEM , Student ' s two - tailed t - Test ) . ( C ) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement . ( Statistics n = 30 - 90 cells , mean ± SEM , * * p < 0 . 001 , * * * p < 0 . 0001 , two - tailed ANOVA . ) ( D ) Representative electron micrographs showing multivesicular endosomal compartments ( arrows ) in control , Clq - and Meth - treated cells at 4 h . Scale bar , 200 nm . ( E ) Ultra - thin cryosections of control , Clq - and Meth - treated dendritic cells as in ( D ) , immunolabeled for LAMP - 1 ( 15 nm gold particles ) and MHC class II ( 10 nm gold particles ) . Scale bar , 200 nm .

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 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 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 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

Figure 1Meth Induces Rapid Alkalization of Acidic Organelles, and Changes in Endosomal Structure in Dendritic Cells(A) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment. Similar results were obtained in three independent experiments. (Statistics n = 25-35, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)(B) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence () or presence (▴) of 50 μM Meth. p < 0.001 for all time points versus control (n = 25-35, mean ± SEM, Student's two-tailed t-Test).(C) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement. (Statistics n = 30-90 cells, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)(D) Representative electron micrographs showing multivesicular endosomal compartments (arrows) in control, Clq- and Meth-treated cells at 4 h. Scale bar, 200 nm.(E) Ultra-thin cryosections of control, Clq- and Meth-treated dendritic cells as in (D), immunolabeled for LAMP-1 (15 nm gold particles) and MHC class II (10 nm gold particles). Scale bar, 200 nm.


Figure 3 ( A ) Accumulation of GFP - LC3 labeled autophagosomes in Meth - or Clq - treated dendritic cells at 2 h and 24 h . Fisher ' s exact test P values versus control ( n = 300 - 400 cells , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 0001 ) . ( B ) GFP - LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment . Similar results were obtained in three independent experiments . Fisher ' s exact test versus control ( n = 300 - 400 cells , mean ± SEM , * * * p < 0 . 0001 ) . ( C ) Representative fluorescence micrographs of GFP - LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h . Arrow denotes examples of autophagosomes . 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 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 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

Figure 3(A) Accumulation of GFP-LC3 labeled autophagosomes in Meth- or Clq-treated dendritic cells at 2 h and 24 h. Fisher's exact test P values versus control (n =300-400 cells, mean ± SEM, *p < 0.05, ***p < 0.0001).(B) GFP-LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment. Similar results were obtained in three independent experiments. Fisher's exact test versus control (n = 300-400 cells, mean ± SEM, ***p < 0.0001).(C) Representative fluorescence micrographs of GFP-LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h. Arrow denotes examples of autophagosomes. Scale bar, 10 μm.


Figure 4 ( A ) Cells were incubated in indicated concentrations of Meth or Clq for 2 h , and then IgG coated erythrocytes were added . Extracellular uningested EIgG were lysed and removed . Phagocytic index ( PI ) was quantified and inhibition is indicated as percent of control . Data were collected from four to seven experiments ( n = 300 cells , mean ± SEM , * * * p < 0 . 0001 , two - tailed ANOVA ) . ( B ) Images of macrophage cells impaired in the phagocytosis of opsinized sheep erythrocytes after 2 h of Meth or Clq treatment . Arrows denote phagocytosed erythrocytes . Scale bar , 10 μm . ( C , D ) J774 . 16 cells were exposed to PBS , chloroquine , or methamphetamine for 2 h followed by incubation with Cn or Ca . The phagocytic indices ( ratio of intracellular yeast to the number of macrophages counted ) were determined after 1 h or 30 min for Cn and Ca , respectively ( C ) . ( Statistics n = 300 , mean ± SEM , * p < 0 . 05 , two - tailed ANOVA . ) CFU determinations were preformed after 24 h incubations ( D ) . The values are the mean and standard deviation from determinations made in triplicate wells ( n = 300 cells , mean ± SEM , * p < 0 . 05 , two - tailed ANOVA . ) . Experiments were repeated three times with similar results .

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 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

Figure 4(A) Cells were incubated in indicated concentrations of Meth or Clq for 2 h, and then IgG coated erythrocytes were added. Extracellular uningested EIgG were lysed and removed. Phagocytic index (PI) was quantified and inhibition is indicated as percent of control. Data were collected from four to seven experiments (n = 300 cells, mean ± SEM, ***p < 0.0001, two-tailed ANOVA).(B) Images of macrophage cells impaired in the phagocytosis of opsinized sheep erythrocytes after 2 h of Meth or Clq treatment. Arrows denote phagocytosed erythrocytes. Scale bar, 10 μm.(C, D) J774.16 cells were exposed to PBS, chloroquine, or methamphetamine for 2 h followed by incubation with Cn or Ca. The phagocytic indices (ratio of intracellular yeast to the number of macrophages counted) were determined after 1 h or 30 min for Cn and Ca, respectively (C). (Statistics n = 300, mean ± SEM, *p < 0.05, two-tailed ANOVA.) CFU determinations were preformed after 24 h incubations (D). The values are the mean and standard deviation from determinations made in triplicate wells (n = 300 cells, mean ± SEM, *p < 0.05, two-tailed ANOVA.). Experiments were repeated three times with similar results.


Figure 1 ( A ) After randomized , 8 - 12 weeks old and sex - matched Phb1F / F mice and Phb1MyeKO mice were administered with PBS or LPS ( 30 mg / kg ) for 12 hours via intraperitoneal injection . Serum was collected and ELISA assays were used to detect the level of IL - 1β ( n = 6 , 10 , 6 , and 12 mice for each group , respectively ) . Data presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( B ) BMDMs were extracted from Phb1F / F or Phb1MyeKO mice and stimulated by 20 % culture supernatant of L929 cells for 7 days . Differentiated BMDMs were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes , the cells were lysed and the culture medium was collected . Western blotting ( WB ) assays were used to detect protein levels in whole cell lysates ( Lys ) and culture medium ( Sup ) . ( C ) BMDMs were extracted from Phb1F / F or Phb1MyeKO mice and stimulated with 20 % culture supernatant of L929 cells for 7 days . Differentiated BMDMs were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . ELISA assays were used to detect protein levels in the culture medium . Data of BMDMs from the separate mice ( n = 6 mice for each group ) and are presented as mean ± SEM . * * * p < 0 . 001 ( Student ' s t - test ) . ( D ) After randomized , 4 weeks old and sex - matched Phb1F / F mice and Phb1MyeKO mice were administered with PBS or LPS ( 300 mg / kg ) to induce septic shock via intraperitoneal injection . The life span of Phb1F / F mice ( n = 6 ) and Phb1MyeKO mice ( n = 7 ) was recorded . ( E ) PBMCs were extracted from normal controls and patients with sepsis ( n = 6 individuals ) . Intracellular mRNA levels were detected by real - time quantitative PCR ( RT - qPCR ) assays ( mean ± SEM ) . n . s . , no significance , * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - test ) . ( F ) PBMCs were extracted from normal controls and infant patients with sepsis . Protein levels in whole cell lysates were detected by WB assays . J774A . 1 cells were stimulated with LPS ( 200 ng / ml ) for 6 hours Intracellular mRNA levels were detected by RT - qPCR assays . Data are from four independent experiments for ( G ) ( mean ± SEM ) . n . s . , no significance , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) . J774A . 1 cells were stimulated with recombinant IL - 1β ( 100 pg / ml ) for 24 hours . Intracellular mRNA levels were detected by RT - qPCR assays . Data are from six independent experiments for ( H ) ( mean ± SEM ) . n . s . , no significance , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( I ) After J774A . 1 cells were stimulated with LPS ( 200 ng / ml ) for 0 , 3 , 6 , 12 hours and ATP ( 4 mM ) for 45 minutes , the cells were lysed , and the culture medium was collected . Protein levels in whole cell lysates ( Lys ) and culture medium ( Sup ) were detected by WB assays . ( J ) After J774A . 1 cells with Phb1 or Phb2 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes , the cells were lysed , and the culture medium was collected . Protein levels in whole cell lysates ( Lys ) and culture medium ( Sup ) were detected by WB assays . ( K ) After J774A . 1 cells with Phb1 or Phb2 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes , the culture medium was collected . ELISA assays were used to detect IL - 1β levels in the culture medium . Data are from three independent experiments ( mean ± SEM ) . n . s . , no significance , * * * * p < 0 . 0001 ( Student ' s t - test ) .

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

Figure 1(A) After randomized, 8-12 weeks old and sex-matched Phb1F/F mice and Phb1MyeKO mice were administered with PBS or LPS (30 mg/kg) for 12 hours via intraperitoneal injection. Serum was collected and ELISA assays were used to detect the level of IL-1β (n = 6, 10, 6, and 12 mice for each group, respectively). Data presented as mean ± SEM. * p < 0.05, ** p < 0.01, **** p< 0.0001 (Student's t-test).(B) BMDMs were extracted from Phb1F/F or Phb1MyeKO mice and stimulated by 20% culture supernatant of L929 cells for 7 days. Differentiated BMDMs were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the cells were lysed and the culture medium was collected. Western blotting (WB) assays were used to detect protein levels in whole cell lysates (Lys) and culture medium (Sup).(C) BMDMs were extracted from Phb1F/F or Phb1MyeKO mice and stimulated with 20% culture supernatant of L929 cells for 7 days. Differentiated BMDMs were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. ELISA assays were used to detect protein levels in the culture medium. Data of BMDMs from the separate mice (n = 6 mice for each group) and are presented as mean ± SEM. *** p < 0.001 (Student's t-test).(D) After randomized, 4 weeks old and sex-matched Phb1F/F mice and Phb1MyeKO mice were administered with PBS or LPS (300 mg/kg) to induce septic shock via intraperitoneal injection. The life span of Phb1F/F mice (n = 6) and Phb1MyeKO mice (n = 7) was recorded.(E) PBMCs were extracted from normal controls and patients with sepsis (n = 6 individuals). Intracellular mRNA levels were detected by real-time quantitative PCR (RT-qPCR) assays (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01 (Student's t-test).(F) PBMCs were extracted from normal controls and infant patients with sepsis. Protein levels in whole cell lysates were detected by WB assays.J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours Intracellular mRNA levels were detected by RT-qPCR assays. Data are from four independent experiments for (G) (mean ± SEM). n.s., no significance, ** p < 0.01, *** p < 0.001, **** p< 0.0001 (Student's t-test).J774A.1 cells were stimulated with recombinant IL-1β (100 pg/ml) for 24 hours. Intracellular mRNA levels were detected by RT-qPCR assays. Data are from six independent experiments for (H) (mean ± SEM). n.s., no significance, ** p < 0.01, *** p < 0.001, **** p< 0.0001 (Student's t-test).(I) After J774A.1 cells were stimulated with LPS (200 ng/ml) for 0, 3, 6, 12 hours and ATP (4 mM) for 45 minutes, the cells were lysed, and the culture medium was collected. Protein levels in whole cell lysates (Lys) and culture medium (Sup) were detected by WB assays.(J) After J774A.1 cells with Phb1 or Phb2 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the cells were lysed, and the culture medium was collected. Protein levels in whole cell lysates (Lys) and culture medium (Sup) were detected by WB assays.(K) After J774A.1 cells with Phb1 or Phb2 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the culture medium was collected. ELISA assays were used to detect IL-1β levels in the culture medium. Data are from three independent experiments (mean ± SEM). n.s., no significance, **** p < 0.0001 (Student's t-test).


Figure 2 ( A - B ) GFP - expressing HeLa cells with stable Phb1 knockdown were fixed and subjected to immunofluorescence ( IF ) analysis to detect mitochondria ( MitoTracker , red ; A ) or ΔΨm ( TMRM , red ; B ) . Images shown are representative of at least three independent experiments . White boxed regions in the panels in ( A ) are enlarged . Scale bar : 10 μm . ( C ) Flow cytometric analysis of ΔΨm ( TMRM ) . HeLa cells with stable Phb1 knockdown were treated with CsA ( 2 μM ) for 30 minutes or FCCP ( 4 μM ) for 5 minutes . ( D ) The mitochondrial respiratory capacity of J774A . 1 cells with or without Phb1 knockdown was measured with a Seahorse XFe96 analyzer . Data are from at least three independent experiments ( mean ± SEM ) . n . s . , no significance . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( E ) HeLa cells with stable PHB1 ablation were incubated with H2O2 ( 2 μM ) and CsA ( 2 μM ) for 30 minutes . Flow cytometric analysis of intracellular ROS levels ( DHE ) . ( F - G ) Confocal microscopy analyses of mitochondrial Ca2 + in time - series mode . HeLa cells stably expressing 4mt - RCaMPh with or without Phb1 knockdown were treated with CsA ( 2 μM ) for 30 minutes . During acquisition of fluorescence images ( F ) , cells were treated with histamine ( 200 μM ) . Traces of mitochondrial Ca2 + ( 4mt - RCaMPh ) dynamics are shown in ( G ) ( mean ± SEM ) . n = 16 , 16 , 15 , 16 ( from the top to the bottom ) . Scale bar : 10 μm . * p < 0 . 05 relative to Scramble group , # p < 0 . 05 relative to shPHB1 group ( Student ' s t - test ) .

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 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 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

Figure 2(A-B) GFP-expressing HeLa cells with stable Phb1 knockdown were fixed and subjected to immunofluorescence (IF) analysis to detect mitochondria (MitoTracker, red; A) or ΔΨm (TMRM, red; B). Images shown are representative of at least three independent experiments. White boxed regions in the panels in (A) are enlarged. Scale bar: 10 μm.(C) Flow cytometric analysis of ΔΨm (TMRM). HeLa cells with stable Phb1 knockdown were treated with CsA (2 μM) for 30 minutes or FCCP (4 μM) for 5 minutes.(D) The mitochondrial respiratory capacity of J774A.1 cells with or without Phb1 knockdown was measured with a Seahorse XFe96 analyzer. Data are from at least three independent experiments (mean ± SEM). n.s., no significance. ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student's t-test).(E) HeLa cells with stable PHB1 ablation were incubated with H2O2 (2 μM) and CsA (2 μM) for 30 minutes. Flow cytometric analysis of intracellular ROS levels (DHE).(F-G) Confocal microscopy analyses of mitochondrial Ca2+ in time-series mode. HeLa cells stably expressing 4mt-RCaMPh with or without Phb1 knockdown were treated with CsA (2 μM) for 30 minutes. During acquisition of fluorescence images (F), cells were treated with histamine (200 μM). Traces of mitochondrial Ca2+ (4mt-RCaMPh) dynamics are shown in (G) (mean ± SEM). n = 16, 16, 15, 16 (from the top to the bottom). Scale bar: 10 μm. * p < 0.05 relative to Scramble group, # p < 0.05 relative to shPHB1 group (Student's t-test).


Figure 3 ( A ) The co - localization between mtDNA ( SG - ALK , green ) and mitochondria ( MitoTracker , red ) was examined in J774A . 1 cells with or without knockdown of Phb1 or Phb2 . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA outside mitochondria . Scale bar : 10 μm . ( B ) Quantitative analysis of data from ( A ) . Data are from ten , eight , and seven images from three independent experiments ( left to right , respectively ) ( mean ± SEM ) . n . s . , no significance , * * * p < 0 . 001 ( Student ' s t - test ) . ( C ) J774A . 1 cells with or without Phb1 knockdown were lysed , and DNA was extracted . qPCR assays were used to detect the mtDNA levels ( D - loop ) in the cytosol relative to nuclear DNA levels ( Tert ) in the whole cell lysates . Data are from six independent experiments ( mean ± SEM ) . * * * * p < 0 . 0001 ( Student ' s t - test ) . ( D ) BMDMs isolated from Phb1F / F mice and Phb1MyeKO mice were fixed and subjected to IF analysis to detect the co - localization between mitochondria ( MitoTracker , red ) and mtDNA ( SG - ALK , green ) . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA outside mitochondria . Scale bar : 10 μm . ( E ) Quantitative analysis of data from ( D ) . Data are from ten and eight images from three independent experiments ( mean ± SEM ) . * * p < 0 . 01 ( Student ' s t - test ) . ( F ) PBMCs were extracted from Phb1F / F mice and Phb1MyeKO mice . Cells were lysed , and DNA was extracted . qPCR assays were used to detect the levels of mtDNA ( D - loop ) in the cytosol relative to the levels of nuclear DNA levels ( Tert ) in the whole cell lysates . Data of PBMC are from seven Phb1F / F mice and Phb1MyeKO mice , respectively ( mean ± SEM ) . * * p < 0 . 01 ( Student ' s t - test ) . ( G ) HeLa cells expressing TFAM - FLAG with or without Phb1 knockdown were incubated with CsA ( 2 μM ) for 30 minutes . Cells were fixed and subjected to IF analysis to detect the co - localization between mitochondria ( anti - TOMM20 , red ) and TFAM - FLAG ( anti - FLAG , green ) . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA outside mitochondria . The yellow arrows indicate mtDNA inside mitochondria . Scale bar : 10 μm . ( H ) Quantitative analysis of data from ( G ) . Data from 30 , 27 , 25 , and 24 images from three independent experiments ( mean ± SEM ) . * * * * p < 0 . 0001 ( Student ' s t - test ) . ( I ) HeLa cells with or without Phb1 knockdown were incubated with H2O2 ( 5 mM ) for 20 minutes and CsA ( 4 μM ) for 12 hours . Cells were lysed , and DNA was extracted . qPCR assays were used to detect mtDNA levels ( ND1 ) in the cytosol and nuclear DNA levels ( 18S ) in the whole cell lysates . Data are from four independent experiments ( mean ± SEM ) . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) . HeLa cells with or without Phb1 knockdown were incubated with VBIT - 4 ( 10 μM ) for 1 hour and subsequently lysed , and DNA was extracted . qPCR assays were used to detect mtDNA levels ( ND1 in HeLa cells in the cytosol relative to nuclear DNA levels ( TERT in HeLa cells in the whole cell lysates . Data are from three independent experiments ( mean ± SEM ) . n . s . , no significance , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Student ' s t - test ) . J774A . 1 cells with or without Phb1 knockdown were incubated with VBIT - 4 ( 10 μM ) for 1 hour and subsequently lysed , and DNA was extracted . qPCR assays were used to detect mtDNA levels D - loop in J774A . 1 cells ) in the cytosol relative to nuclear DNA levels Tert in J774A . 1 cells ) in the whole cell lysates . Data are from three independent experiments ( mean ± SEM ) . n . s . , no significance , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Student ' s t - test ) . ( L ) J774A . 1 cells with or without Phb1 knockdown were lysed . The whole cell lysates were immunoprecipitated by IgG or anti - ASC antibody and the resulting proteins were detected by WB assay . ( M ) BMDMs isolated from Phb1F / F mice and Phb1MyeKO mice were incubated with MCC950 ( 50 nM ) for 30 mins and were subsequently stimulated by LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . Protein levels in whole cell lysates and culture medium were detected by WB assay .

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 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 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 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 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 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 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 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

Figure 3(A) The co-localization between mtDNA (SG-ALK, green) and mitochondria (MitoTracker, red) was examined in J774A.1 cells with or without knockdown of Phb1 or Phb2. White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar: 10 μm. (B) Quantitative analysis of data from (A). Data are from ten, eight, and seven images from three independent experiments (left to right, respectively) (mean ± SEM). n.s., no significance, *** p < 0.001 (Student's t-test).(C) J774A.1 cells with or without Phb1 knockdown were lysed, and DNA was extracted. qPCR assays were used to detect the mtDNA levels (D-loop) in the cytosol relative to nuclear DNA levels (Tert) in the whole cell lysates. Data are from six independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(D) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were fixed and subjected to IF analysis to detect the co-localization between mitochondria (MitoTracker, red) and mtDNA (SG-ALK, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar: 10 μm. (E) Quantitative analysis of data from (D). Data are from ten and eight images from three independent experiments (mean ± SEM). ** p < 0.01 (Student's t-test).(F) PBMCs were extracted from Phb1F/F mice and Phb1MyeKO mice. Cells were lysed, and DNA was extracted. qPCR assays were used to detect the levels of mtDNA (D-loop) in the cytosol relative to the levels of nuclear DNA levels (Tert) in the whole cell lysates. Data of PBMC are from seven Phb1F/F mice and Phb1MyeKO mice, respectively (mean ± SEM). ** p < 0.01 (Student's t-test).(G) HeLa cells expressing TFAM-FLAG with or without Phb1 knockdown were incubated with CsA (2 μM) for 30 minutes. Cells were fixed and subjected to IF analysis to detect the co-localization between mitochondria (anti-TOMM20, red) and TFAM-FLAG (anti-FLAG, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. The yellow arrows indicate mtDNA inside mitochondria. Scale bar: 10 μm.(H) Quantitative analysis of data from (G). Data from 30, 27, 25, and 24 images from three independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(I) HeLa cells with or without Phb1 knockdown were incubated with H2O2 (5 mM) for 20 minutes and CsA (4 μM) for 12 hours. Cells were lysed, and DNA was extracted. qPCR assays were used to detect mtDNA levels (ND1) in the cytosol and nuclear DNA levels (18S) in the whole cell lysates. Data are from four independent experiments (mean ± SEM). ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student's t-test).HeLa cells with or without Phb1 knockdown were incubated with VBIT-4 (10 μM) for 1 hour and subsequently lysed, and DNA was extracted. qPCR assays were used to detect mtDNA levels (ND1 in HeLa cells in the cytosol relative to nuclear DNA levels (TERT in HeLa cells in the whole cell lysates. Data are from three independent experiments (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student's t-test).J774A.1 cells with or without Phb1 knockdown were incubated with VBIT-4 (10 μM) for 1 hour and subsequently lysed, and DNA was extracted. qPCR assays were used to detect mtDNA levels D-loop in J774A.1 cells) in the cytosol relative to nuclear DNA levels Tert in J774A.1 cells) in the whole cell lysates. Data are from three independent experiments (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student's t-test).(L) J774A.1 cells with or without Phb1 knockdown were lysed. The whole cell lysates were immunoprecipitated by IgG or anti-ASC antibody and the resulting proteins were detected by WB assay.(M) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were incubated with MCC950 (50 nM) for 30 mins and were subsequently stimulated by LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. Protein levels in whole cell lysates and culture medium were detected by WB assay.


Figure 4 ( A ) BMDMs isolated from Phb1F / F mice and Phb1MyeKO mice were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . Cells were fixed and subjected to IF analysis to detect the co - localization between NLRP3 ( anti - NLRP3 , red ) and mtDNA ( PicoGreen , green ) . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA co - localized with NLRP3 . Scale bar : 10 μm . ( B ) Quantitative analysis of data from ( A ) . Data are from at least 14 images from three independent experiments ( mean ± SEM ) . * * * * p < 0 . 0001 ( Student ' s t - test ) . ( C ) J774A . 1 cells with or without Phb1 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . Cells were fixed and subjected to IF analysis to detect the co - localization between NLRP3 ( anti - NLRP3 , red ) and mtDNA ( PicoGreen , green ) . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA co - localized with NLRP3 . Scale bar : 10 μm . ( D ) Quantitative analysis of data from ( C ) . Data are from at least six images from three independent experiments ( mean ± SEM ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( E ) J774A . 1 cells with or without Phb1 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . After cells were lysed , NLRP3 was immunoprecipitated by anti - NLRP3 antibody . mtDNA levels ( D - loop ) from immunoprecipitants were detected by qPCR in J774A . 1 cells with or without Phb1 knockdown . Data are from five independent experiments ( mean ± SEM ) . * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( F ) J774A . 1 cells with Phb1 knockdown were incubated with EtBr ( 150 ng / ml ) for 3 days , followed by LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . WB was used to detect the protein levels in whole cell lysates and culture medium . ( G ) BMDMs isolated from Phb1F / F mice and Phb1MyeKO mice were treated with NAC ( 2 mM ) for 8 hours , LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . WB assay was used to detect the protein levels in whole cell lysates and in the culture medium .

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 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 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 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 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

Figure 4(A) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. Cells were fixed and subjected to IF analysis to detect the co-localization between NLRP3 (anti-NLRP3, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA co-localized with NLRP3. Scale bar: 10 μm. (B) Quantitative analysis of data from (A). Data are from at least 14 images from three independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(C) J774A.1 cells with or without Phb1 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. Cells were fixed and subjected to IF analysis to detect the co-localization between NLRP3 (anti-NLRP3, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA co-localized with NLRP3. Scale bar: 10 μm.(D) Quantitative analysis of data from (C). Data are from at least six images from three independent experiments (mean ± SEM). * p <0.05, ** p < 0.01, **** p < 0.0001 (Student's t-test).(E) J774A.1 cells with or without Phb1 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. After cells were lysed, NLRP3 was immunoprecipitated by anti-NLRP3 antibody. mtDNA levels (D-loop) from immunoprecipitants were detected by qPCR in J774A.1 cells with or without Phb1 knockdown. Data are from five independent experiments (mean ± SEM). ** p < 0.01, **** p < 0.0001 (Student's t-test).(F) J774A.1 cells with Phb1 knockdown were incubated with EtBr (150 ng/ml) for 3 days, followed by LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB was used to detect the protein levels in whole cell lysates and culture medium.(G) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were treated with NAC (2 mM) for 8 hours, LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and in the culture medium.


Figure 5 ( A ) HeLa cells were incubated with H2O2 ( 5 mM ) for 20 minutes and then lysed . The whole cell lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . WB was used to detect the SPG7 or AFG3L2 levels , respectively , in the precipitated products . ( B ) J774A . 1 cells were stimulated with LPS ( 200 ng / ml ) for 6 hours and lysed . The whole cell lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . The respective levels of precipitated SPG7 or AFG3L2 were examined by WB . HeLa cells with or without Phb1 knockdown were lysed . The whole cell lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . The respective levels of precipitated SPG7 or AFG3L2 were examined by WB . J774A . 1 cells with or without Phb1 knockdown were lysed . The whole cell lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . The respective levels of precipitated SPG7 or AFG3L2 were examined by WB .

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 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 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

Figure 5(A) HeLa cells were incubated with H2O2 (5 mM) for 20 minutes and then lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. WB was used to detect the SPG7 or AFG3L2 levels, respectively, in the precipitated products.(B) J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours and lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. The respective levels of precipitated SPG7 or AFG3L2 were examined by WB.HeLa cells with or without Phb1 knockdown were lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. The respective levels of precipitated SPG7 or AFG3L2 were examined by WB.J774A.1 cells with or without Phb1 knockdown were lysed. The whole cell lysate was immunoprecipitated by IgG, anti-AFG3L2 antibody or anti-SPG7 antibody. The respective levels of precipitated SPG7 or AFG3L2 were examined by WB.


Figure 6 ( A ) J774A . 1 cells with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were fixed and subjected to IF analysis to assess the co - localization between mitochondria ( MitoTracker , red ) and mtDNA ( PicoGreen , green ) . White boxed regions in the panels are enlarged . The white arrows indicate mtDNA outside mitochondria . Scale bar : 10 μm . ( B ) Quantitative analysis of the data from ( A ) . Data are from at least 12 images from three independent experiments ( mean ± SEM ) . * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - test ) . ( C ) J774A . 1 cells with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were lysed , and DNA was extracted . qPCR was used to detect the mtDNA levels ( D - loop ) in the cytosol relative to the nuclear DNA levels ( Tert ) in the whole cell lysate . Data are from six independent experiments ( mean ± SEM ) . * * * * p < 0 . 0001 ( Student ' s t - test ) . ( D ) J774A . 1 cells with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . The culture medium was collected and ELISA assays were used to detect IL - 1β levels in culture medium . Data are from four independent experiments ( mean ± SEM ) . * * * * p < 0 . 0001 ( Student ' s t - test ) . ( E ) J774A . 1 cells with Phb1 knockdown , Spg7 knockdown , or combined Phb1 and Spg7 knockdowns were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . WB assay was used to detect the protein levels in whole cell lysates and culture medium . ( F ) J774A . 1 cells with Phb1 knockdown , Afg3l2 knockdown , and combined Phb1 and Afg3l2 knockdowns were stimulated with LPS ( 200 ng / ml ) for 6 hours and ATP ( 4 mM ) for 45 minutes . WB assay was used to detect the protein levels in whole cell lysates and culture medium . ( G ) J774A . 1 cells were stimulated with LPS ( 200 ng / ml ) for 6 hours and lysed . The whole cell lysate was immunoprecipitated by IgG or anti - SPG7 antibody . WB was used to detect the CYPD and VDAC levels in the precipitated products . ( H ) J774A . 1 cells with or without Phb1 knockdown were lysed . The whole cell lysates were immunoprecipitated by IgG or anti - SPG7 antibody . WB was used to detect the CYPD and VDAC levels in the precipitated products .

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 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 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 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 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 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

Figure 6(A) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were fixed and subjected to IF analysis to assess the co-localization between mitochondria (MitoTracker, red) and mtDNA (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar: 10 μm. (B) Quantitative analysis of the data from (A). Data are from at least 12 images from three independent experiments (mean ± SEM). ** p < 0.01, **** p < 0.0001 (Student's t-test).(C) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were lysed, and DNA was extracted. qPCR was used to detect the mtDNA levels (D-loop) in the cytosol relative to the nuclear DNA levels (Tert) in the whole cell lysate. Data are from six independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(D) J774A.1 cells with knockdown of Phb1 alone, Afg3l2 alone, Spg7 alone, Phb1 + Afg3l2, or Phb1 + Spg7 were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. The culture medium was collected and ELISA assays were used to detect IL-1β levels in culture medium. Data are from four independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(E) J774A.1 cells with Phb1 knockdown, Spg7 knockdown, or combined Phb1 and Spg7 knockdowns were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and culture medium.(F) J774A.1 cells with Phb1 knockdown, Afg3l2 knockdown, and combined Phb1 and Afg3l2 knockdowns were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. WB assay was used to detect the protein levels in whole cell lysates and culture medium.(G) J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours and lysed. The whole cell lysate was immunoprecipitated by IgG or anti-SPG7 antibody. WB was used to detect the CYPD and VDAC levels in the precipitated products.(H) J774A.1 cells with or without Phb1 knockdown were lysed. The whole cell lysates were immunoprecipitated by IgG or anti-SPG7 antibody. WB was used to detect the CYPD and VDAC levels in the precipitated products.


Figure 1B Representative pictures of different CTCs from the initial CellSearch ® analysis of the metastatic breast cancer patient who gave rise to the breast CTC line . The detected tumor cells display clear keratin and DAPI staining , CD45 - negativity as well as lack of , or very weak ( 4 , 8 ) , ERBB2 expression . Cells of small ( about 5 µm in diameter , 1 , 2 ) and large size ( larger than 10µm in diameter , 2 , 3 ) were detected . While some CTCs displayed dot - like perinuclear keratin signals ( 1 , 2 ) , the majority showed diffuse keratin staining . Additionally , CTC clusters of 4 - 8 cells were present ( 5 , 6 ) . Some CTCs showed multiple / lobed nuclei ( 4 , 7 , 8 ) . C Bright field images of CTC - ITB - 01 cells growing adherently . D Bright field images of CTC - ITB - 01 cells growing non - adherently ( relation between adherent and non - adherent cells : 80 / 20 % ) .

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

Figure 1B Representative pictures of different CTCs from the initial CellSearch® analysis of the metastatic breast cancer patient who gave rise to the breast CTC line. The detected tumor cells display clear keratin and DAPI staining, CD45-negativity as well as lack of, or very weak (4, 8), ERBB2 expression. Cells of small (about 5 µm in diameter, 1, 2) and large size (larger than 10µm in diameter, 2,3) were detected. While some CTCs displayed dot-like perinuclear keratin signals (1, 2), the majority showed diffuse keratin staining. Additionally, CTC clusters of 4-8 cells were present (5, 6). Some CTCs showed multiple/lobed nuclei (4, 7, 8).C Bright field images of CTC-ITB-01 cells growing adherently. D Bright field images of CTC-ITB-01 cells growing non-adherently (relation between adherent and non-adherent cells: 80/20 %).


Figure 2Unsupervised hierarchical clustering ( Ward linkage with Euclidean distance ) of CNA profiles generated from patient primary tumors , the vaginal metastasis , isolated single and pooled CTCs from the primary blood collection and CTC - ITB - 01 ( early and later passage ) and leukocytes . " CL " represents the cell line , followed by the passage analyzed ( p04 / p30 ) . " CTC " marks circulating tumor cells isolated from the initial blood draw , followed by 01 / 05 / 10 indicating single ( 01 ) or pooled cells ( 5 or 10 , respectively ) . Light and dark grey bars to the left signify chromosomes , starting from the top with chromosome 1 . Red color represents chromosome loss , blue represents gain . All copy number aberrations were calculated using the patient ' s own germline DNA extracted from leukocytes as reference to eliminate copy number variations ( CNVs ) .

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

Figure 2Unsupervised hierarchical clustering (Ward linkage with Euclidean distance) of CNA profiles generated from patient primary tumors, the vaginal metastasis, isolated single and pooled CTCs from the primary blood collection and CTC-ITB-01 (early and later passage) and leukocytes. "CL" represents the cell line, followed by the passage analyzed (p04/p30). "CTC" marks circulating tumor cells isolated from the initial blood draw, followed by 01/05/10 indicating single (01) or pooled cells (5 or 10, respectively). Light and dark grey bars to the left signify chromosomes, starting from the top with chromosome 1. Red color represents chromosome loss, blue represents gain. All copy number aberrations were calculated using the patient's own germline DNA extracted from leukocytes as reference to eliminate copy number variations (CNVs).


Figure 3Direct comparisons of CNA profiles . Percentages of gene copy number gains and losses across the chromosomes for different sample types . Copy number gains are displayed in green , losses in red . A CNAs generated from CTCs from the primary blood collection ( upper panel ) and CTC - ITB - 01 , indicated as " CL " ( medium panel ) . Significant distinctions between both samples were calculated using Fisher ' s exact test ( Joosse et al , 2018 ) and are indicated in lowest panel ( blue ) . Increased blue color intensity represents lower p - value and therefore higher statistical significance . Direct comparisons of CNA profiles . Percentages of gene copy number gains and losses across the chromosomes for different sample types . Copy number gains are displayed in green , losses in red . B CNA comparison between early ( p04 ) and late ( p30 ) CTC - ITB - 01 passages ( upper and medium panel ) . Significant distinctions between both samples were calculated using Fisher ' s exact test ( Joosse et al , 2018 ) and are indicated in lowest panel ( in blue ) . Increased blue color intensity represents lower p - value and therefore higher statistical significance .

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

Figure 3Direct comparisons of CNA profiles. Percentages of gene copy number gains and losses across the chromosomes for different sample types. Copy number gains are displayed in green, losses in red. A CNAs generated from CTCs from the primary blood collection (upper panel) and CTC-ITB-01, indicated as "CL" (medium panel). Significant distinctions between both samples were calculated using Fisher's exact test (Joosse et al, 2018) and are indicated in lowest panel (blue). Increased blue color intensity represents lower p-value and therefore higher statistical significance.Direct comparisons of CNA profiles. Percentages of gene copy number gains and losses across the chromosomes for different sample types. Copy number gains are displayed in green, losses in red. B CNA comparison between early (p04) and late (p30) CTC-ITB-01 passages (upper and medium panel). Significant distinctions between both samples were calculated using Fisher's exact test (Joosse et al, 2018) and are indicated in lowest panel (in blue). Increased blue color intensity represents lower p-value and therefore higher statistical significance.


Figure 4A Western blot analysis of selected protein markers , including ERα , EGFR , ERBB2 , EpCAM , K18 , K19 , K8 , E - cadherin , N - cadherin , vimentin , CD44 , CD24 , SNAIL , SLUG , TWIST and α - tubulin ( as a loading control ) ( n = 3 replicates ) . CTC - ITB - 01 was compared to more mesenchymal ER - Hs578t and epithelial ER + MCF - 7 breast cancer cell lines . B ICC staining of CTC - ITB - 01 for pan - keratin ( orange ) , ER ( green ) , and DAPI ( blue ) . The scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 ( 1 ) and ( 2 ) . MCF - 7 cells are depicted as reference cell line . C PAM50 classifier results showing probabilities of pooled CTC - ITB - 01 cells matching specific molecular breast cancer subtypes . Starting from lowest probability , CTC - ITB - 01 was classified as 1 . 02 % ( s = 2 . 5 % ) normal - like , 3 . 11 % ( s = 6 . 6 % ) Basal - like , 13 . 11 % ( s = 9 . 3 % ) ERBB2 - positive , 16 . 77 % ( s = 23 . 7 % ) luminal A , and 65 . 22 % ( s = 16 . 6 % ) luminal B breast cancer subtype . Data was generated from n = 3 replicates . D PAM50 classifier results showing probabilities of the non - adherent and adherent CTC - ITB - 01 fractions matching molecular subtypes . Selected bars are not visible due to extremely low probability ( close to zero ) . Both fractions show greatest alignment with a luminal B subtype . Starting from lowest probability , the adherent fraction of CTC - ITB - 01 was classified as 0 % ( ± 0 % ) normal - like , 6 . 22 % ( ± 9 . 0 % ) Basal - like , 17 . 85 % ( ± 8 . 6 % ) ERBB2 - positive , 0 % ( s = 0 % ) luminal A , and 75 . 92 % ( s = 0 . 7 % ) luminal B breast cancer subtype . The suspension cell fraction of CTC - ITB - 01 was classified as 2 . 04 % ( s = 3 . 5 % ) normal - like , 0 % ( s = 0 % ) Basal - like , 17 . 32 % ( s = 9 . 7 % ) ERBB2 - positive , 33 . 5 % ( s = 23 . 8 % ) luminal A , and 68 . 67 % ( s = 18 . 6 % ) luminal B breast cancer subtype , respectively . Data was generated from n = 3 replicates . ( E ) ALDH activity measurement on viable cells via flow cytometry using the ALDEFLUOR ™ assay . CTC - ITB - 01 is compared to A549 as recommended control cell line . An internal ( DEAB ) control is also depicted . 77 . 92 % of the gated CTC - ITB - 01 population are ALDH + .

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 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 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 O O O O O O O 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 4A Western blot analysis of selected protein markers, including ERα, EGFR, ERBB2, EpCAM, K18, K19, K8, E-cadherin, N-cadherin, vimentin, CD44, CD24, SNAIL, SLUG, TWIST and α-tubulin (as a loading control) (n=3 replicates). CTC-ITB-01 was compared to more mesenchymal ER- Hs578t and epithelial ER+ MCF-7 breast cancer cell lines.B ICC staining of CTC-ITB-01 for pan-keratin (orange), ER (green), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01 (1) and (2). MCF-7 cells are depicted as reference cell line.C PAM50 classifier results showing probabilities of pooled CTC-ITB-01 cells matching specific molecular breast cancer subtypes. Starting from lowest probability, CTC-ITB-01 was classified as 1.02% (s=2.5%) normal-like, 3.11% (s=6.6%) Basal-like, 13.11% (s=9.3%) ERBB2-positive, 16.77% (s=23.7%) luminal A, and 65.22% (s=16.6%) luminal B breast cancer subtype. Data was generated from n=3 replicates.D PAM50 classifier results showing probabilities of the non-adherent and adherent CTC-ITB-01 fractions matching molecular subtypes. Selected bars are not visible due to extremely low probability (close to zero). Both fractions show greatest alignment with a luminal B subtype. Starting from lowest probability, the adherent fraction of CTC-ITB-01 was classified as 0% (±0%) normal-like, 6.22% (±9.0%) Basal-like, 17.85% (±8.6%) ERBB2-positive, 0% (s=0%) luminal A, and 75.92% (s=0.7%) luminal B breast cancer subtype. The suspension cell fraction of CTC-ITB-01 was classified as 2.04% (s=3.5%) normal-like, 0% (s=0%) Basal-like, 17.32% (s=9.7%) ERBB2-positive, 33.5% (s=23.8%) luminal A, and 68.67% (s=18.6%) luminal B breast cancer subtype, respectively. Data was generated from n=3 replicates.(E) ALDH activity measurement on viable cells via flow cytometry using the ALDEFLUOR™ assay. CTC-ITB-01 is compared to A549 as recommended control cell line. An internal (DEAB) control is also depicted. 77.92% of the gated CTC-ITB-01 population are ALDH+.