( A ) showing UHMK1 , p - NCOA3 ( S1062 / T1067 ) , ATIC expression in two human GC specimens . Scale bars = 100μm . ( B ) Pearson correlation about of of UHMK1 , p - NCOA3 ( S1062 / T1067 ) ATIC in with GC ( n = 120 ) by .

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(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 stratified by level of UHMK1 . ( Cii ) Combination of UHMK1 p - NCOA3 ( S1062 / T1067 ) expression predicts more exact OS of GC . Survival curves were plotted according to Kaplan - Meier method , with log - rank applied for comparison .

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(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.


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

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D 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.


D , E PS in LUVs enhances 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 western blot band density of oligomers / ( monomer + dimer ) from chemical crosslinking experiments . A one - way ANOVA was performed with multiple comparisons compared to 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 .

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D, 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.


D , E of PS clustering in HEK293 in response to fendiline treatment through N & B . D Left panel : Representative from time - lapse ( 30 frames ) imaging of HEK293 expressing GFP - LactC2 treated with fendiline for 48 hours ; scale bar = 5 µm . Middle panel : Brightness plots for each representative image . Right panel : Selection map correlating each pixel in representative image to an oligomerization state ( b value ) ( red : monomer - 5mer , green : 5mer - 10mer , blue : > 10mer ) . E Average % pixels from panel ( d ) = of GFP - LactC2 with brightness values corresponding to monomer - 5mer ( ~ 1 . - 1 . 5 ) , 5mer - 10mer ( ~ 1 . 5 - 1 . 9 ) > 10mer ( > 1 . 9 ) over 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 control DMSO % average pixels ( * * * * p < 0 . 0001 , * * p = 0 . 0043 ) . Data information : GFP - LactC2 : phosphatidylserine sensor ; N & B : Number & Brightness ; PS : phosphatidylserine .

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D, 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.


Effect of fendiline on EBOV infection . A Representative confocal of Vero E6 infected with EBOV ( Kikwit ) at indicated MOI treated with indicated of fendiline . were pretreated 24 hours prior to infection with indicated of fendiline . Post infection , were treated 1 hour later ( d - 1 / 0 ) , treated every day ( e . d ) , or treated every other day ( e . o . d ) fixed at either 48 hours , 72 hours or 96 hours post infection . ( green = EBOV ; blue = nuclei ) . numbering in top right corner indicates % infection . Scale bar = 100 μm . Data information : EBOV : Ebola ; MOI : multiplicity of

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Effect 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 infection


Effect of fendiline on EBOV infection . B - D 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 ; MOI : multiplicity of infection ; d . - 1 / 0 : treatment 1 hour after infection ; e . d . : treatment every day ; e . o . d . : treatment every other day .

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Effect 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.


A - C Effect of fendiline on eVP40 mVP40 PM localization in HEK293 after 48 hours of treatment . A Representative confocal from live imaging experiments of HEK293 expressing EGFP - WT - eVP40 ( top panel ) EGFP - WT - mVP40 ( bottom panel ) after 48 hours of fendiline treatment . scale bars = 10 µm . Effect of fendiline on mVP40 ( B ) eVP40 ( C ) PM localization was quantified by of EGFP at PM / total EGFP ( normalized to DMSO control ) . N > 15 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 DMSO control . Data information : PM : plasma

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A-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 membrane


D , E of eVP40 oligomerization in HEK293 in response to 48 hour fendiline treatment using N & B . D Left panel : Representative from time - lapse ( 30 frames ) of HEK293 expressing EGFP - WT - eVP40 treated with fendiline for 48 hours . scale bar = 5 µm . Middle panel : Brightness plots for each representative image . Right panel : Selection map correlating each pixel in representative image to an oligomerization state ( b value ) ( red : monomer - hexamer , green : hexamer - 12mer , blue : 12mer - 24mer , pink : > 24mer ) . E Average % pixel 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 ) > 24mer ( > 3 . 2 ) over total pixels within each image . Values are reported as mean ± s . d . ; N ≥ 9 per biological replicate , n = 3 biological replicates ; A two - way ANOVA was performed with Dunnett ' s multiple comparisons compared to control DMSO % average pixels ( * * p = 0 . 0035 ) . Data information : N & B : Number & Brightness .

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D, 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.


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

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

A-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.


A - C of eVLP . A Representative transmission electron of eVLPs purified from HEK293 expressing FLAG - eVP40 eGP following 48 hours of DMSO ( left panel ) or 5 µM fendiline treatment ( right panel ) . B of eVLP ( µm ) of DMSO - derived eVLPs ( black ) fendiline - derived eVLPs ( blue ) . N > 50 VLPs per biological replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A two - t - was performed ( * * p = 0 . 0139 ) . C of eVLP diameter ( nm ) of DMSO - derived eVLPs ( black ) fendiline - derived eVLPs ( blue ) . N > 50 VLPs per biological replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A two - t - was performed ( * p = 0 . 0430 ) . Data information : : transmission electron microscopy ; eVLPs : entry - competent VLPs ; eGP : Ebola

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A-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 glycoprotein


D , E DiI TIM - 1 dependent entry . D Representative confocal from DiI - entry comparing entry of eVLPs produced from DMSO ( top panel ) fendiline - treated HEK293 ( bottom panel ) into target ( HEK293 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 of eVLP entry was performed by calculating total number of DiI punctate / total number of DiI - positive . Three from each z - stack were quantified . N = 9 per replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons against 0 . 0 µg TIM - 1 condition for both DMSO - fendiline derived eVLPs . ( * * * * p < 0 . 0001 ; * * p = 0 . 0093 ) . Data information : TIM - 1 : t - immunoglobulin receptor - 1 ; eVLPs : entry - competent VLPs ; DiI : 1 , 1 ' - Dioctadecyl - 3 , 3 , 3 ' , 3 ' - Tetramethylindocarbocyanine Perchlorate .

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D, 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.


B . Before differentiation , hESCs show uniformly high expression of OCT4 . Treatment with BMP4 produces a mixture of OCT4 + / CDX2 - self - renewing hESCs OCT4 - / CDX2 + mesodermal .

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B. 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 . of OCT4 CDX2 expression by immunofluorescence after 24 h of BMP4 treatment reveals two populations of hESCs . were assigned to one of two distinct populations on a 2 - component mixed Gaussian

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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 distribution


E . Filmstrip of OCT4 dynamics in an undifferentiated hESC throughout its cycle duration . Yellow outlines indicate region used to quantify mean .

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E. 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 in hESCs . A single OCT4 level was quantified for each by averaging mean mCherry over lifetime of .

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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 - traces of OCT4 signaling . of each ' s trace indicates its cycle duration .

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G. Single-cell traces of OCT4 signaling. The length of each cell's trace indicates its cell cycle duration.


H . Distribution of cycle durations for 120 hESCs .

H. Distribution of cell cycle durations for 120 hESCs.


A . Single - traces of hESCs before after treatment with 100 ng / mL BMP4 . were imaged for 42 h prior to BMP4 treatment . Mean OCT4 were quantified every 5 minutes were tracked from division event that created until its own division .

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A. 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 , were fixed , stained for expression of CDX2 , returned to microscope for registration with final time - lapse image . Mean OCT4 - mCherry CDX2 were quantified for each , resulting distribution was fit to a 2 - component mixed Gaussian distribution representing self - renewing ( OCT4 + / CDX2 - ) differentiated ( OCT4 - / CDX2 + ) hESCs that could be assigned to either distribution with > 99 % confidence ( red green dots , Pdiff < 0 . 01 or Pdiff > 0 . 99 ) were considered for pro - fate . that did not reach this threshold ( gray dots ) were not used to determine pro - fate . Because in final frame were assigned to only self - renewing or differentiated categories , pro - mixed

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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 cells


C . Distributions of OCT4 in pro - self - renewing , pro - mixed , pro - differentiated populations .

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C. Distributions of OCT4 levels in pro-self-renewing, pro-mixed, and pro-differentiated cell populations.


D . Distributions of OCT4 frequencies in pro - pluripotent , pro - mixed , pro - differentiated populations . To gain an unbiased look at preexisting determinants of fate in panels only who completed their entire cycle duration before BMP4 addition ( t = 0 ) were included in .

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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 cycle durations in pro - pluripotent , pro - mixed , pro - differentiated populations . To gain an unbiased look at preexisting determinants of fate in panels , only who completed their entire cycle duration before BMP4 addition ( t = 0 ) were included in .

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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 . with self - renewing ( red ) or differentiated ( green ) pro - fates are plotted above predicted class surfaces . ( left ) Slice of decision surface for frequency vs . OCT4 at a cycle duration of 14 h . ( right ) Slice of decision surface for cycle duration vs . OCT4 level at a frequency of 0 . 25 h - 1 . * P < 0 . 05 , * * P < 0 . 0005 ; ns , not significant .

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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.


A . Lineage of OCT4 expression dynamics . Mean OCT4 were quantified in hESCs continuously for 72 h under undifferentiated conditions . Vertical bars represent that completed an entire cycle lifetime . Thin horizontal bars denote division events . Color scale indicates low ( black ) , intermediate ( red ) , high ( ) OCT4 expression .

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A. 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 between sister , cousin , second cousin , randomly paired .

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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 during division .

C. Filmstrip showing distribution of OCT4 to daughter cells during cell division.


D . Distribution of OCT4 ratios between sister before after BMP4 treatment . Ratios for both sister ( r 1 / r ) are plotted to emphasize central tendency toward r = 1 : 1 . Differently colored curves represent distribution of ratios at different time points after division . For time points after 5 minutes , was determined by first calculating a mean OCT4 level for each sister among previous time points then calculating resulting between sisters .

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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 , stained for content with DAPI . probability density of DAPI ( blue ) OCT4 - mCherry ( red ) between sister that divided ≤ 1 h prior to fixation was calculated for 151 pairs using a normal kernel function . A two - sample Kolmogorov - Smirnov was used to determine significance . * P < 0 . 05 , * * * P < 0 . 0005 , two - sample Kolmogorov - Smirnov ; ns , not significant .

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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.


B . OCT4 mixing as a function of history . 30 randomly chosen were colored according to their OCT4 expression level at 8 h after division .

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B. 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 , autonomous periods of history . At each time point , coefficient of determination ( R2 ) between total OCT4 expression final OCT4 expression at 8 h was calculated .

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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 . same R2 over time mother - daughter traces were randomly sampled from a set of mother - daughter traces that contained sister - pairs ( blue ) or only one randomly selected sister from each pair ( red ) . E . Difference in variance explained between red blue traces in panel D . black represents actual difference ; red shows a smoothing ( moving average = 1 . 25 h ) of black trace to highlight overall trend .

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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.


Meth Induces Rapid Alkalization of Acidic Organelles , Changes in Endosomal Structure in Dendritic ( A ) Morphometric of decrease in acridine orange after 10 min treatment . Similar results were obtained in three independent experiments . ( Statistics n = 25 - 35 , mean ± , * * p < 0 . 001 , * * * p < 0 . 0001 , two - ANOVA . )

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Meth 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 acridine orange over 4 h in absence ( ) or presence ( ▴ ) of 50 μM Meth . p < 0 . 001 for time points versus control ( n = 25 - 35 , mean ± , Student ' s two - t - ) .

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(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 exposed to Meth for 10 min measured by ratiometric measurement . ( Statistics n = 30 - 90 , mean ± , * * p < 0 . 001 , * * * p < 0 . 0001 , two - ANOVA . )

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(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 showing multivesicular endosomal compartments ( arrows ) in control , Clq - Meth - treated at 4 h . Scale bar , 200 nm .

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(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 - Meth - treated dendritic as in ( D ) , immunolabeled for LAMP - 1 ( 15 nm gold particles ) MHC class II ( 10 nm gold particles ) . Scale bar , 200 nm .

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(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.


( A ) Accumulation of GFP - LC3 labeled autophagosomes in Meth - or Clq - treated dendritic at 2 h 24 h . Fisher ' s exact P values versus control ( n = 300 - 400 , mean ± , * p < 0 . 05 , * * * p < 0 . 0001 ) .

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(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 versus control ( n = 300 - 400 , mean ± , * * * p < 0 . 0001 ) .

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(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 of GFP - LC3 decorated autophagosomes in treated with 50 μM Meth or 10 μM Clq for 24 h . Arrow denotes examples of autophagosomes . Scale bar , 10 μm .

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(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.


( A ) were incubated in indicated concentrations of Meth or Clq for 2 h , then IgG coated erythrocytes were added . Extracellular uningested EIgG were lysed removed . Phagocytic index ( PI ) was quantified inhibition is indicated as of control . Data were collected from four to seven experiments ( n = 300 , mean ± , * * * p < 0 . 0001 , two - ANOVA ) .

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(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 ) of macrophage impaired in phagocytosis of opsinized sheep erythrocytes after 2 h of Meth or Clq treatment . Arrows denote phagocytosed erythrocytes . Scale bar , 10 μm .

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(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 were exposed to PBS , chloroquine , or methamphetamine for 2 h followed by incubation with Cn or Ca . phagocytic indices ( of intracellular yeast to number of macrophages counted ) were determined after 1 h or 30 min for Cn Ca , respectively ( C ) . ( Statistics n = 300 , mean ± , * p < 0 . 05 , two - ANOVA . ) CFU determinations were preformed after 24 h incubations ( D ) . values are mean standard deviation from determinations made in triplicate wells ( n = 300 , mean ± , * p < 0 . 05 , two - ANOVA . ) . Experiments were repeated three times with similar results .

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(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.


( A ) After randomized , 8 - 12 old sex - matched Phb1F / F mice Phb1MyeKO mice were administered with PBS or LPS ( 30 mg / kg ) for 12 hours via intraperitoneal injection . Serum was collected ELISA assays were used to detect level of IL - 1β ( n = 6 , 10 , 6 , 12 mice for each group , respectively ) . Data presented as mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - ) .

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(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 stimulated by 20 % culture supernatant of L929 for 7 days . Differentiated BMDMs were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes , were lysed culture medium was collected . Western blotting ( WB ) assays were used to detect in whole lysates ( Lys ) culture medium ( Sup ) .

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(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 stimulated with 20 % culture supernatant of L929 for 7 days . Differentiated BMDMs were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . ELISA assays were used to detect in culture medium . Data of BMDMs from separate mice ( n = 6 mice for each group ) are presented as mean ± . * * * p < 0 . 001 ( Student ' s t - ) .

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(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 old sex - matched Phb1F / F mice Phb1MyeKO mice were administered with PBS or LPS ( 300 mg / kg ) to induce septic shock via intraperitoneal injection . life span of Phb1F / F mice ( n = 6 ) Phb1MyeKO mice ( n = 7 ) was recorded .

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(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 with sepsis ( n = 6 individuals ) . Intracellular mRNA were detected by real - time quantitative PCR ( RT - qPCR ) assays ( mean ± ) . n . s . , no significance , * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - ) .

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(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 infant with sepsis . in whole lysates were detected by WB assays .

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(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 were stimulated with LPS ( 200 ng / ml ) for 6 hours Intracellular mRNA were detected by RT - qPCR assays . Data are from four independent experiments for ( G ) ( mean ± ) . n . s . , no significance , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - ) .

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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 were stimulated with recombinant IL - 1β ( 100 pg / ml ) for 24 hours . Intracellular mRNA were detected by RT - qPCR assays . Data are from six independent experiments for ( H ) ( mean ± ) . n . s . , no significance , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - ) .

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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 were stimulated with LPS ( 200 ng / ml ) for 0 , 3 , 6 , 12 hours ATP ( 4 mM ) for 45 minutes , were lysed , culture medium was collected . in whole lysates ( Lys ) culture medium ( Sup ) were detected by WB assays .

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(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 with Phb1 or Phb2 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes , were lysed , culture medium was collected . in whole lysates ( Lys ) culture medium ( Sup ) were detected by WB assays .

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

(J) 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 with Phb1 or Phb2 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes , culture medium was collected . ELISA assays were used to detect IL - 1β in culture medium . Data are from three independent experiments ( mean ± ) . n . s . , no significance , * * * * p < 0 . 0001 ( Student ' s t - ) .

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

(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).


( A - B ) GFP - expressing HeLa with stable Phb1 knockdown were fixed subjected to immunofluorescence ( IF ) to detect mitochondria ( MitoTracker , red ; A ) or ΔΨm ( TMRM , red ; B ) . shown are representative of at least three independent experiments . boxed regions in panels in ( A ) are enlarged . Scale bar : 10 μm .

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

(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 of ΔΨm ( TMRM ) . HeLa with stable Phb1 knockdown were treated with CsA ( 2 μM ) for 30 minutes or FCCP ( 4 μM ) for 5 minutes .

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

(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 ) mitochondrial respiratory capacity of J774A . 1 with or without Phb1 knockdown was measured with a Seahorse XFe96 analyzer . Data are from at least three independent experiments ( mean ± ) . n . s . , no significance . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - ) .

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

(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 with stable PHB1 ablation were incubated with H2O2 ( 2 μM ) CsA ( 2 μM ) for 30 minutes . Flow cytometric of intracellular ROS ( DHE ) .

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(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 stably expressing 4mt - RCaMPh with or without Phb1 knockdown were treated with CsA ( 2 μM ) for 30 minutes . During acquisition of ( F ) , were treated with histamine ( 200 μM ) . Traces of mitochondrial Ca2 + ( 4mt - RCaMPh ) dynamics are shown in ( G ) ( mean ± ) . n = 16 , 16 , 15 , 16 ( from top to bottom ) . Scale bar : 10 μm . * p < 0 . 05 relative to Scramble group , # p < 0 . 05 relative to shPHB1 group ( Student ' s t - ) .

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

(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).


( A ) co - localization between mtDNA ( SG - ALK , green ) mitochondria ( MitoTracker , red ) was examined in J774A . 1 with or without knockdown of Phb1 or Phb2 . boxed regions in panels are enlarged . arrows indicate mtDNA outside mitochondria . Scale bar : 10 μm . ( B ) Quantitative of data from ( A ) . Data are from ten , eight , seven from three independent experiments ( left to right , respectively ) ( mean ± ) . n . s . , no significance , * * * p < 0 . 001 ( Student ' s t - ) .

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

(A) 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 with or without Phb1 knockdown were lysed , was extracted . qPCR assays were used to detect mtDNA ( D - loop ) in cytosol relative to ( Tert ) in whole lysates . Data are from six independent experiments ( mean ± ) . * * * * p < 0 . 0001 ( Student ' s t - ) .

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

(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 Phb1MyeKO mice were fixed subjected to IF to detect co - localization between mitochondria ( MitoTracker , red ) mtDNA ( SG - ALK , green ) . boxed regions in panels are enlarged . arrows indicate mtDNA outside mitochondria . Scale bar : 10 μm . ( E ) Quantitative of data from ( D ) . Data are from ten eight from three independent experiments ( mean ± ) . * * p < 0 . 01 ( Student ' s t - ) .

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

(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 Phb1MyeKO mice . were lysed , was extracted . qPCR assays were used to detect of mtDNA ( D - loop ) in cytosol relative to of ( Tert ) in whole lysates . Data of PBMC are from seven Phb1F / F mice Phb1MyeKO mice , respectively ( mean ± ) . * * p < 0 . 01 ( Student ' s t - ) .

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

(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 expressing TFAM - FLAG with or without Phb1 knockdown were incubated with CsA ( 2 μM ) for 30 minutes . were fixed subjected to IF to detect co - localization between mitochondria ( anti - TOMM20 , red ) TFAM - FLAG ( anti - FLAG , green ) . boxed regions in panels are enlarged . arrows indicate mtDNA outside mitochondria . yellow arrows indicate mtDNA inside mitochondria . Scale bar : 10 μm .

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

(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 of data from ( G ) . Data from 30 , 27 , 25 , 24 from three independent experiments ( mean ± ) . * * * * p < 0 . 0001 ( Student ' s t - ) .

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

(H) 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 with or without Phb1 knockdown were incubated with H2O2 ( 5 mM ) for 20 minutes CsA ( 4 μM ) for 12 hours . were lysed , was extracted . qPCR assays were used to detect mtDNA ( ND1 ) in cytosol ( 18S ) in whole lysates . Data are from four independent experiments ( mean ± ) . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - ) .

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

(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 with or without Phb1 knockdown were incubated with VBIT - 4 ( 10 μM ) for 1 hour subsequently lysed , was extracted . qPCR assays were used to detect mtDNA ( ND1 in HeLa in cytosol relative to ( TERT in HeLa in whole lysates . Data are from three independent experiments ( mean ± ) . n . s . , no significance , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Student ' s t - ) .

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

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 with or without Phb1 knockdown were incubated with VBIT - 4 ( 10 μM ) for 1 hour subsequently lysed , was extracted . qPCR assays were used to detect mtDNA D - loop in J774A . 1 ) in cytosol relative to Tert in J774A . 1 ) in whole lysates . Data are from three independent experiments ( mean ± ) . n . s . , no significance , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Student ' s t - ) .

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

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 with or without Phb1 knockdown were lysed . whole lysates were immunoprecipitated by IgG or anti - ASC antibody resulting were detected by WB .

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

(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 Phb1MyeKO mice were incubated with MCC950 ( 50 nM ) for 30 mins were subsequently stimulated by LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . in whole lysates culture medium were detected by WB .

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

(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.


( A ) BMDMs isolated from Phb1F / F mice Phb1MyeKO mice were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . were fixed subjected to IF to detect co - localization between NLRP3 ( anti - NLRP3 , red ) mtDNA ( PicoGreen , green ) . boxed regions in panels are enlarged . arrows indicate mtDNA co - localized with NLRP3 . Scale bar : 10 μm . ( B ) Quantitative of data from ( A ) . Data are from at least 14 from three independent experiments ( mean ± ) . * * * * p < 0 . 0001 ( Student ' s t - ) .

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

(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 with or without Phb1 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . were fixed subjected to IF to detect co - localization between NLRP3 ( anti - NLRP3 , red ) mtDNA ( PicoGreen , green ) . boxed regions in panels are enlarged . arrows indicate mtDNA co - localized with NLRP3 . Scale bar : 10 μm .

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

(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 of data from ( C ) . Data are from at least six from three independent experiments ( mean ± ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - ) .

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

(D) 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 with or without Phb1 knockdown were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . After were lysed , NLRP3 was immunoprecipitated by anti - NLRP3 antibody . mtDNA ( D - loop ) from immunoprecipitants were detected by qPCR in J774A . 1 with or without Phb1 knockdown . Data are from five independent experiments ( mean ± ) . * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - ) .

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

(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 with Phb1 knockdown were incubated with EtBr ( 150 ng / ml ) for 3 days , followed by LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . WB was used to detect in whole lysates culture medium .

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

(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 Phb1MyeKO mice were treated with NAC ( 2 mM ) for 8 hours , LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . WB was used to detect in whole lysates in culture medium .

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

(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.


( A ) HeLa were incubated with H2O2 ( 5 mM ) for 20 minutes then lysed . whole lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . WB was used to detect SPG7 or AFG3L2 , respectively , in precipitated products .

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

(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 were stimulated with LPS ( 200 ng / ml ) for 6 hours lysed . whole lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . respective of precipitated SPG7 or AFG3L2 were examined by WB .

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

(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 with or without Phb1 knockdown were lysed . whole lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . respective of precipitated SPG7 or AFG3L2 were examined by WB .

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

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 with or without Phb1 knockdown were lysed . whole lysate was immunoprecipitated by IgG , anti - AFG3L2 antibody or anti - SPG7 antibody . respective of precipitated SPG7 or AFG3L2 were examined by WB .

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

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.


( A ) J774A . 1 with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were fixed subjected to IF to assess co - localization between mitochondria ( MitoTracker , red ) mtDNA ( PicoGreen , green ) . boxed regions in panels are enlarged . arrows indicate mtDNA outside mitochondria . Scale bar : 10 μm . ( B ) Quantitative of data from ( A ) . Data are from at least 12 from three independent experiments ( mean ± ) . * * p < 0 . 01 , * * * * p < 0 . 0001 ( Student ' s t - ) .

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

(A) 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 with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were lysed , was extracted . qPCR was used to detect mtDNA ( D - loop ) in cytosol relative to ( Tert ) in whole lysate . Data are from six independent experiments ( mean ± ) . * * * * p < 0 . 0001 ( Student ' s t - ) .

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(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 with knockdown of Phb1 alone , Afg3l2 alone , Spg7 alone , Phb1 + Afg3l2 , or Phb1 + Spg7 were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . culture medium was collected ELISA assays were used to detect IL - 1β in culture medium . Data are from four independent experiments ( mean ± ) . * * * * p < 0 . 0001 ( Student ' s t - ) .

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(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 with Phb1 knockdown , Spg7 knockdown , or combined Phb1 Spg7 knockdowns were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . WB was used to detect in whole lysates culture medium .

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(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 with Phb1 knockdown , Afg3l2 knockdown , combined Phb1 Afg3l2 knockdowns were stimulated with LPS ( 200 ng / ml ) for 6 hours ATP ( 4 mM ) for 45 minutes . WB was used to detect in whole lysates culture medium .

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(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 were stimulated with LPS ( 200 ng / ml ) for 6 hours lysed . whole lysate was immunoprecipitated by IgG or anti - SPG7 antibody . WB was used to detect CYPD VDAC in precipitated products .

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(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 with or without Phb1 knockdown were lysed . whole lysates were immunoprecipitated by IgG or anti - SPG7 antibody . WB was used to detect CYPD VDAC in precipitated products .

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(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.


B Representative pictures of different CTCs from initial CellSearch ® of metastatic breast cancer who gave rise to breast CTC . detected display clear keratin DAPI , CD45 - negativity as well as lack of , or very weak ( 4 , 8 ) , ERBB2 expression . of small ( about 5 µm in diameter , 1 , 2 ) large ( larger than 10µm in diameter , 2 , 3 ) were detected . While some CTCs displayed dot - like perinuclear keratin signals ( 1 , 2 ) , majority showed diffuse keratin . Additionally , CTC clusters of 4 - 8 were present ( 5 , 6 ) . Some CTCs showed multiple / lobed nuclei ( 4 , 7 , 8 ) .

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B 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 of CTC - ITB - 01 growing adherently . D Bright field of CTC - ITB - 01 growing non - adherently ( relation between adherent non - adherent : 80 / 20 % ) .

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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 %).


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

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Unsupervised 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).


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

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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. 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 losses across chromosomes for different sample types . Copy number gains are displayed in green , losses in red . B CNA comparison between early ( p04 ) late ( p30 ) CTC - ITB - 01 passages ( upper medium panel ) . Significant distinctions between both samples were calculated using Fisher ' s exact ( Joosse et al , 2018 ) are indicated in lowest panel ( in blue ) . Increased blue color represents lower p - value therefore higher statistical significance .

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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.


A Western blot of selected markers , including ERα , EGFR , ERBB2 , EpCAM , K18 , K19 , K8 , E - cadherin , N - cadherin , vimentin , CD44 , CD24 , SNAIL , SLUG , TWIST α - tubulin ( as a loading control ) ( n = 3 replicates ) . CTC - ITB - 01 was compared to more mesenchymal ER - Hs578t epithelial ER + MCF - 7 breast cancer lines .

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A 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 of CTC - ITB - 01 for pan - keratin ( orange ) , ER ( green ) , DAPI ( blue ) . scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 ( 1 ) ( 2 ) . MCF - 7 are depicted as reference .

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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 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 , 65 . 22 % ( s = 16 . 6 % ) luminal B breast cancer subtype . Data was generated from n = 3 replicates .

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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 non - adherent 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 , 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 , 75 . 92 % ( s = 0 . 7 % ) luminal B breast cancer subtype . suspension 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 , 68 . 67 % ( s = 18 . 6 % ) luminal B breast cancer subtype , respectively . Data was generated from n = 3 replicates .

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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 via flow cytometry using ALDEFLUOR ™ . CTC - ITB - 01 is compared to A549 as recommended control . An internal ( DEAB ) control is also depicted . 77 . 92 % of gated CTC - ITB - 01 population are ALDH + .

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(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+.


A ICC of CTC - ITB - 01 for ß - catenin ( green ) , E - cadherin ( orange ) , DAPI ( blue ) . scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 .

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A ICC staining of CTC-ITB-01 for ß-catenin (green), E-cadherin (orange), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.


B ICC of CTC - ITB - 01 for α - catenin ( green ) , visualization of actin filaments by phalloidin ( orange ) , nuclei by DAPI ( blue ) . scale bar corresponds to 20 µm . Two representative panels are shown for CTC - ITB - 01 .

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B ICC staining of CTC-ITB-01 for α-catenin (green), visualization of actin filaments by phalloidin (orange), and nuclei by DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.


C Generic EMT score of adherent non - adherently growing CTC - ITB - 01 fractions in relation to samples from different cancer entities . Scaling ranges from 1 ( completely epithelial ) to - 1 ( entirely mesenchymal ) . adherent fraction is shown with a black dot , non - adherent fraction with a black triangle , mean score per cancer entity by a black . D Generic EMT score of adherent non - adherently growing CTC - ITB - 01 fractions in relation to established cancer lines . Scaling ranges from 1 ( completely epithelial ) to - 1 ( entirely mesenchymal ) . adherent fraction is shown with a black dot , non - adherent fraction with a black triangle , mean score per cancer entity by a black .

C Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to tumor samples from different cancer entities. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line. D Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to established cancer cell lines. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line.


A In vivo bioluminescence imaging of CTC - ITB - 01 in four NSG mice . Blue indicates low radiance , red indicates high radiance . B Logarithmic plotting of in vivo radiance increase over time . Curves represent means ± of measurements performed on multiple ( n = 8 ) .

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A In vivo bioluminescence imaging of CTC-ITB-01 in four NSG mice. Blue indicates low radiance, red indicates high radiance. B Logarithmic plotting of in vivo radiance increase over time. Curves represent means ± SEM of measurements performed on multiple glands (n=8).


C H & E of xenografted mouse mammary show formation of tumors .

C H&E staining of xenografted mouse mammary glands show formation of primary tumors.


D Stereoscope image of a mammary of immunodeficient NSG mouse . Fluorescently tagged CTC - ITB - 01 in green .

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D Stereoscope image of a mammary gland of immunodeficient NSG mouse. Fluorescently tagged CTC-ITB-01 cells in green.


E for ER , confirms retained in vivo ER - positivity of formed tumors . Representative of ER in higher ( left panel ) lower ( right panel ) magnification of 2 different areas of a xenografted mammary .

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E IHC staining for ER, confirms retained in vivo ER-positivity of formed tumors. Representative images of ER staining in higher (left panel) and lower (right panel) magnification of 2 different areas of a xenografted mammary gland.


F Representative ex vivo luminescence of indicated organs from engrafted mice . Organs include brain ( 1 ) , lung ( 2 ) , liver ( 3 ) bones ( 4 ) . G Scatter blot showing ex vivo radiance of metastatic organs in three different mice ( M1 , M2 , M3 ) , indicating highest in liver bone . Data represent means ± SD of measurements performed on metastatic organs derived from three different mice .

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F Representative ex vivo luminescence images of indicated organs from engrafted mice. Organs include brain (1), lung (2), liver (3) and bones (4). G Scatter blot showing ex vivo radiance intensity of metastatic organs in three different mice (M1, M2, M3), indicating highest intensity in liver and bone. Data represent means ± SD of measurements performed on metastatic organs derived from three different mice.