GSEA of consensus squamous cancer marker genes ( se Appendix Table S2 ) in A - 431 cells stably transduced with sh - ∆ Np63 # 2 or sh - NTC . ( N ) ES : ( normalised ) enrichment score GSEA of consensus squamous cancer marker genes in A - 431 cells stably transduced with sh - USP28 # 1 or sh - NTC . ( N ) ES : ( normalised ) enrichment

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

GSEA of consensus squamous cancer marker genes (se Appendix Table S2) in A-431 cells stably transduced with sh-∆Np63#2 or sh-NTC. (N)ES: (normalised) enrichment score GSEA of consensus squamous cancer marker genes in A-431 cells stably transduced with sh-USP28#1 or sh-NTC. (N)ES: (normalised) enrichment score


Correlation of mRNA expression of consensus squamous cancer marker genes and TP63 in lung SCC and non - transformed lung tissue ( Normal ) . R : Spearmans correlation coefficient . n = 836 . Gepia software . Correlation of mRNA expression of consensus squamous cancer marker genes and USP28 in lung SCC and non - transformed lung tissue ( Normal ) . R : Spearmans correlation coefficient . N = 836 . Gepia software .

Correlation of mRNA expression of consensus squamous cancer marker genes and TP63 in lung SCC and non-transformed lung tissue (Normal). R: Spearmans correlation coefficient. n=836. Gepia software. Correlation of mRNA expression of consensus squamous cancer marker genes and USP28 in lung SCC and non-transformed lung tissue (Normal). R: Spearmans correlation coefficient. N=836. Gepia software.


Representative haematoxylin and eosin ( H & E ) staining of tumour bearing animals 12 weeks post intratracheal infection . Boxes indicate highlighted tumour areas in ( C ) ( a , b , a ' and b ' ) . Scale bar = 2000µm ; nKPL = 6 and nKPLU = 5 . H = heart .

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

Representative haematoxylin and eosin (H&E) staining of tumour bearing animals 12 weeks post intratracheal infection. Boxes indicate highlighted tumour areas in (C) (a, b, a' and b'). Scale bar = 2000µm; nKPL= 6 and nKPLU = 5. H=heart.


Representative IHC staining for ADC ( TTF - 1 ) and SCC ( KRT5 and ∆ Np63 ) marker expression as well as Usp28 abundance in KPL ( n = 6 ) and KPLU ( n = 5 ) lung tumours . Scale bar : 20µm .

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

Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 abundance in KPL (n=6) and KPLU (n=5) lung tumours. Scale bar: 20µm.


Quantification of % tumour area ( normalised to total lung area ) in KPL ( n = 6 ) and KPLU ( n = 5 ) animals . Quantification of lung tumour numbers in KPL ( n = 6 ) and KPLU ( n = 5 ) animals .

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

Quantification of % tumour area (normalised to total lung area) in KPL (n=6) and KPLU (n=5) animals. Quantification of lung tumour numbers in KPL (n=6) and KPLU (n=5) animals.


Immunoblot of endogenous LKB1 , USP28 and ∆ Np63 in KPL and KPLU tumours . ( VINCULIN as loading control ) . Western blot shown are representative of three independent experiments ( n = 3 ) . Representative immunoblot of endogenous NOTCH1 , c - MYC and c - JUN in KPL and KPLU ( ACTIN as a control ) . n =

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

Immunoblot of endogenous LKB1, USP28 and ∆Np63 in KPL and KPLU tumours. (VINCULIN as loading control). Western blot shown are representative of three independent experiments (n=3). Representative immunoblot of endogenous NOTCH1, c-MYC and c-JUN in KPL and KPLU (ACTIN as a control). n=3


Kaplan - Meier survival curves comparing KPL ( n = 6 ) and KPLU ( n = 5 ) animals upon AAV intratracheal infection . The p - value was calculated using a log - rank test .

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

Kaplan-Meier survival curves comparing KPL (n=6) and KPLU (n=5) animals upon AAV intratracheal infection. The p-value was calculated using a log-rank test.


Representative qPCR of SCC and ADC marker expression in two independent KP lung tumour clones , resulting in KPADC and KPSCC ; ( ACTIN served as loading control ) . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) .

Representative qPCR of SCC and ADC marker expression in two independent KP lung tumour clones, resulting in KPADC and KPSCC; (ACTIN served as loading control). Quantitative graph is represented as mean ± SD of three experiments (n=3).


Immunoblot against endogenous USP28 in the murine KPSCC cell line upon targeting with two independent shRNA sequences . ACTIN served as loading control .

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

Immunoblot against endogenous USP28 in the murine KPSCC cell line upon targeting with two independent shRNA sequences. ACTIN served as loading control.


Representative haematoxylin and eosin ( H & E ) images of tumour bearing animals 8 weeks post intratracheal transplantation of 2 × 105 cells / animal . KPSCC sh - NTC ( n = 3 ) ; KPSCC sh - USP28 ( n = 3 ) , Scale bar =

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

Representative haematoxylin and eosin (H&E) images of tumour bearing animals 8 weeks post intratracheal transplantation of 2 × 105 cells/animal. KPSCC sh-NTC (n=3); KPSCC sh-USP28 (n=3), Scale bar = 5000µm


Quantification of % tumour area ( top , normalised to total lung area ) and lung tumour numbers ( bottom ) on KPSCC sh - NTC ( n = 6 ) and KPSCC sh - USP28 ( n = 6 ) animals .

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

Quantification of % tumour area (top, normalised to total lung area) and lung tumour numbers (bottom) on KPSCC sh-NTC (n=6) and KPSCC sh-USP28 (n=6) animals.


Representative IHC staining for ADC ( TTF - 1 ) and SCC ( KRT5 and ∆ Np63 ) marker expression as well as Usp28 and GFP abundance in KPSCC sh - NTC ( n = 3 ) and KPSCC sh - USP28 ( n = 3 ) lung tumours . Scale bar = 50

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

Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 and GFP abundance in KPSCC sh-NTC (n=3) and KPSCC sh-USP28 (n=3) lung tumours. Scale bar =50 µm


Analysis of occurring TP63 genetic alterations in lung squamous ( LUSC ) , cervical ( CESC ) , oesophagus ( ESCA ) , head and neck ( HNSC ) and pancreatic ( PAAD ) tumours . Cbioportal .

Analysis of occurring TP63 genetic alterations in lung squamous (LUSC), cervical (CESC), oesophagus (ESCA), head and neck (HNSC) and pancreatic (PAAD) tumours. Cbioportal.


Expression of TP63 ( left ) and USP28 ( right ) in human Lung ( n = 498 ) , Cervix ( n = 254 ) , Oesophagus ( n = 96 ) and HNSC ( n = 522 ) SCC tumours and normal non - transformed tissue ( nLung = 338 nCervix = 3 , nOesophagus = 11 and nHNSC = 44 ) . In box plots , the centre line reflects the median , the cross represents the mean and the upper and lower box limits indicates the first and third quartile . Whiskers extend 1 . 5x the IQR . p - values were calculated using two - tailed t - test statistical analysis . Xena UCSC software .

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

Expression of TP63 (left) and USP28 (right) in human Lung (n=498), Cervix (n=254), Oesophagus (n=96) and HNSC (n=522) SCC tumours and normal non-transformed tissue (nLung=338 nCervix=3, nOesophagus=11 and nHNSC=44). In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR. p-values were calculated using two-tailed t-test statistical analysis. Xena UCSC software.


Correlation of mRNA expression of KRT14 / KRT7 and TP63 in ADC and SCC tumours for Lung ( nADC = 513 and nSCC = 498 ) , Cervix ( nADC = 47 and nSCC = 254 ) and Oesophagus ( nADC = 89 and nSCC = 96 ) . Blue dots : ADC ; Red dots : SCC ; R = Spearmans correlation coefficient ; m : Slope . Xena UCSC .

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

Correlation of mRNA expression of KRT14 / KRT7 and TP63 in ADC and SCC tumours for Lung (nADC=513 and nSCC=498), Cervix (nADC=47 and nSCC=254) and Oesophagus (nADC=89 and nSCC=96). Blue dots: ADC; Red dots: SCC; R= Spearmans correlation coefficient; m: Slope. Xena UCSC.


Immunoblot of control ( sh - NTC ) and two independent shRNA targeting USP28 ( sh - USP28 # 1 and # 2 ) for ∆ Np63 , KRT14 and USP28 protein abundance in H1299 , LUDLU - 1adh , HELA , SiHa , Ca Ski , PANC - 1 and BXPC - 3 ( ACTIN as loading control ) .

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

Immunoblot of control (sh-NTC) and two independent shRNA targeting USP28 (sh-USP28#1 and #2) for ∆Np63, KRT14 and USP28 protein abundance in H1299, LUDLU-1adh, HELA, SiHa, Ca Ski, PANC-1 and BXPC-3 (ACTIN as loading control).


Cells were seeded at equal cell density and counted after five days , Brigth field images of control or sh - USP28 # 2 infected H1299 , LUDLU - 1adh , Hela , CaSKI , PANC - 1 and BXPC - 3 cells before quantification . Scale bar = 30µm Relative number of H1299 , LUDLU - 1adh , Hela , CaSKI , SiHa , PANC - 1 and BXPC - 3 sh - USP28 # 2 cells compared with sh - NTC control cells . p - values were calculated using two - tailed t - test statistical analysis . SiHa * = Notably , the human SCC cell line SiHa was negative for ΔNp63 .

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

Cells were seeded at equal cell density and counted after five days, Brigth field images of control or sh-USP28#2 infected H1299, LUDLU-1adh, Hela, CaSKI, PANC-1 and BXPC-3 cells before quantification. Scale bar = 30µm Relative number of H1299, LUDLU-1adh, Hela, CaSKI, SiHa, PANC-1 and BXPC-3 sh-USP28#2 cells compared with sh-NTC control cells. p-values were calculated using two-tailed t-test statistical analysis. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63.


Immunoblot of endogenous USP28 in A - 431 cells upon treatment with warheads ( WH ) and either DMSO or indicated concentrations of AZ1 ( USP28 Inh . ) for 24 hours . USP28 Upper band : Active USP28 ; USP28 lower band : Inactive USP28 , VINCULIN as loading control .

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

Immunoblot of endogenous USP28 in A-431 cells upon treatment with warheads (WH) and either DMSO or indicated concentrations of AZ1 (USP28 Inh.) for 24 hours. USP28 Upper band: Active USP28; USP28 lower band: Inactive USP28, VINCULIN as loading control.


Immunoblot of endogenous USP28 , ∆ Np63 and KRT14 in A - 431 cells treated for 24 hours with either DMSO or indicated concentrations of AZ1 . VINCULIN served as loading control . TP63 and USP28 half maximal inhibitory protein abundance ( IC50 ) was calculated . Graphs are represented as mean ± SD ( n = 3 ) .

O 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

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control. TP63 and USP28 half maximal inhibitory protein abundance (IC50) was calculated. Graphs are represented as mean ± SD (n=3).


TUBE pulldown of endogenous ubiquitylated ∆ Np63 in A - 431 cells upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours . VINCULIN served as loading control for relative Ubiquitination .

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

TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours. VINCULIN served as loading control for relative Ubiquitination.


Immunoblot of endogenous USP28 , ∆ Np63 and KRT14 in A - 431 cells treated for 18 hours with either DMSO or 15µM AZ1 followed by 6 hours of treatment with 20µM proteasomal inhibitor MG - 132 ( 20µM ) or DMSO . VINCULIN served as loading control .

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

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 18 hours with either DMSO or 15µM AZ1 followed by 6 hours of treatment with 20µM proteasomal inhibitor MG-132 (20µM) or DMSO. VINCULIN served as loading control.


A - 431 cells were seeded at equal cell density and cultured in the presence of either DMSO , 1µM , 10µM or 30µM AZ1 for 48h . Cells were quantified with the Operetta imaging system using Hoechst staining . 50 % growth inhibition ( GI50 ) was calculated . Scale bar = 250µm . n = 30 fields

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

A-431 cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. Scale bar = 250µm. n= 30 fields analysed


Immunoblot of endogenous USP28 , ∆ Np63 and KRT14 in LUDLU - 1adh , H1299 , Ca Ski , Hela and SiHa cells treated for 24 hours with either DMSO or indicated concentrations of AZ1 . VINCULIN served as loading

O O O O O O O O O O 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

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in LUDLU-1adh, H1299, Ca Ski, Hela and SiHa cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control


LUDLU - 1ADH , H1299 , Ca Ski , Hela and SiHa cells were seeded at equal cell density and cultured in the presence of either DMSO , 1µM , 10µM or 30µM AZ1 for 48h . Scale bar for LUDLU - 1adh and H1299 = 500µm . Scale bar for Ca Ski , Hela and SiHa = 250µm . SiHa * = Notably , the human SCC cell line SiHa was negative for ΔNp63 . Number of cells were quantified with the Operetta imaging system using Hoechst staining . 50 % growth inhibition ( GI50 ) was calculated . n = 30 fields

O O O O O O O O O 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

LUDLU-1ADH, H1299, Ca Ski, Hela and SiHa cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Scale bar for LUDLU-1adh and H1299 = 500µm. Scale bar for Ca Ski, Hela and SiHa = 250µm. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63. Number of cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. n=30 fields analysed


Immunofluorescence ( IF ) staining against endogenous ∆ Np63 in KP and KPL cell lines ( blue : DAPI [ staining of nuclei ] and red / magenta : ∆ Np63 ) , Scale bar = 32µm .

Immunofluorescence (IF) staining against endogenous ∆Np63 in KP and KPL cell lines (blue: DAPI [staining of nuclei] and red/magenta: ∆Np63), Scale bar = 32µm.


GFP Fluorescent images of lungs treated with either PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . Tumour cells are positive for GFP ( green arrows ) . n = 3 Quantification of % tumour area ( normalised to total lung area ) in control ( n = 5 ) , 125 mg / kg AZ1 ( n = 4 ) and 375 mg / kg AZ1 ( n = 4 ) treated animals .

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

GFP Fluorescent images of lungs treated with either PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Tumour cells are positive for GFP (green arrows). n=3 Quantification of % tumour area (normalised to total lung area) in control (n=5), 125 mg/kg AZ1 (n=4) and 375 mg/kg AZ1 (n=4) treated animals.


Representative staining of H & E and IHC for USP28 and ∆ Np63 of SCC tumours from mice treated with PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . Boxes indicate highlighted tumour areas . First line Scale bars = 500µm ; Lower scale bars = 20µm . n = 3 ∆ Np63 staining intensity of lung tumours treated with PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . nControl = 33712 cells ; nAZ1 125mg / kg = 7382 cells ; nAZ1 375mg / kg = 4967 cells . Cells were analysed from three independent animals .

O O O O O O O O O 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 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

Representative staining of H&E and IHC for USP28 and ∆Np63 of SCC tumours from mice treated with PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Boxes indicate highlighted tumour areas. First line Scale bars = 500µm; Lower scale bars = 20µm. n=3   ∆Np63 staining intensity of lung tumours treated with PBS/DMSO/0,1%Tween80 or indicated concentrations of AZ1. nControl = 33712 cells ; nAZ1 125mg/kg = 7382 cells; nAZ1 375mg/kg = 4967 cells. Cells were analysed from three independent animals.


A Western blot analysis STAT expression and phosphorylation . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 250 IU / mL of IFNβ or 5 ng / mL of IFNγ for 0 . 5 , 6 , 12 and 24 h . Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 ( Y701 ) and STAT2 ( Y689 ) and total level of STAT1 and STAT2 . The blots are representative of more than 3 independent experiments .

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

A Western blot analysis STAT expression and phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ for 0.5, 6, 12 and 24 h. Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 (Y701) and STAT2 (Y689) and total level of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.


B Effect of STAT1Y701Fheterozygosity on the expression of type I IFN - induced genes ( ISG ) . BMDMs of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 250 IU / mL of IFNβ for 4 and 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . The bars represent mean values with the standard deviations ( SD ) of three independent experiments . C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ - induced genes . BMDMs of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 5 ng / mL of IFNγ for 4 and 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . The bars represent mean values with the standard deviations ( SD ) of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Effect of STAT1Y701Fheterozygosity on the expression of type I IFN-induced genes (ISG). BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ-induced genes. BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.


A Effect of STAT1Y701F homozygosity on STAT1 expression in mouse cells and organs . Spleens , livers or bone marrow derived macrophages ( BMDMs ) were isolated from wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice . Whole cell extracts were collected and tested for levels of total Stat1 in western blot . The blots are representative of more than 3 independent experiments .

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

A Effect of STAT1Y701F homozygosity on STAT1 expression in mouse cells and organs. Spleens, livers or bone marrow derived macrophages (BMDMs) were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice. Whole cell extracts were collected and tested for levels of total Stat1 in western blot. The blots are representative of more than 3 independent experiments.


B Effect of STAT1Y701F homozygosity on STAT1 phosphorylation at Y701 . BMDMs were isolated from wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice and stimulated for 30 min with 250 IU / mL of IFNβ or 5 ng / mL of IFNγ . Whole cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in western blot . The blots are representative of more than 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Effect of STAT1Y701F homozygosity on STAT1 phosphorylation at Y701. BMDMs were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice and stimulated for 30 min with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ. Whole cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in western blot. The blots are representative of more than 3 independent experiments.


C Effect of STAT1Y701Fhomozygosity on the expression of IFNβ - induced genes . BMDMs of wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were treated with 5 ng / mL of IFNγ for 4 or 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( ns , p > 0 . 05 ) ; the p - values were calculated using paired ratio t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Effect of STAT1Y701Fhomozygosity on the expression of IFNβ-induced genes. BMDMs of wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05); the p-values were calculated using paired ratio t-test.


D Effect of STAT1Y701Fhomozygosity on the expression of type I IFN - induced genes ( ISG ) . BMDMs were isolated from wild type ( WT ) , Stat1Y701F , Stat1 - / - , Stat2 - / - and IRF9 - / - mice treated with 250 IU / mL of IFNβ for 4 , 8 , 12 , 24 or 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( ns , p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ) ; the p - values were calculated using paired ratio t - test .

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D Effect of STAT1Y701Fhomozygosity on the expression of type I IFN-induced genes (ISG). BMDMs were isolated from wild type (WT), Stat1Y701F, Stat1-/-, Stat2-/- and IRF9-/- mice treated with 250 IU/mL of IFNβ for 4, 8, 12, 24 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05; *, p≤ 0.05; **, p≤ 0.01); the p-values were calculated using paired ratio t-test.


E STAT1 and STAT2 phosphorylation in Stat1 - / - , Stat1Y701F , Stat2 - / - and Irf9 - / - macrophages . BMDMs were isolated from wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice and treated with 250 IU / mL of IFNβ for 30 min or 6 , 12 or 24 h . The whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689 . The same cell extracts were tested for total levels of STAT1 and STAT2 . The blots are representative of more than 3 independent experiments .

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E STAT1 and STAT2 phosphorylation in Stat1-/-, Stat1Y701F, Stat2-/- and Irf9-/- macrophages. BMDMs were isolated from wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice and treated with 250 IU/mL of IFNβ for 30 min or 6, 12 or 24 h. The whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689. The same cell extracts were tested for total levels of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.


A IFNβ - stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with STAT2 - specific antibody . The amount of precipitated DNA was measured by Q - PCR . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( * * , p ≤ 0 . 01 ) ; the p - values were calculated using paired ratio t - test .

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A IFNβ-stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT2-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (**, p≤ 0.01); the p-values were calculated using paired ratio t-test.


B Impact of STAT2 deficiency on IFNβ - stimulated STAT1 association with the Mx2 ISRE . BMDMs of wild type ( WT ) , Stat1 - / - ( S1 ) and Stat2 - / - ( S2 ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with STAT1 - specific antibody . The amount of precipitated DNA was measured by Q - PCR . Bars represent mean values of three independent experiments ; error bars represent standard error of mean ( SEM ) .

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B Impact of STAT2 deficiency on IFNβ -stimulated STAT1 association with the Mx2 ISRE. BMDMs of wild type (WT), Stat1-/- (S1) and Stat2-/- (S2) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT1-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard error of mean (SEM).


C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP - reChIP . BMDMs of wild type ( WT ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with either STAT1 - specific antibody and re - immunoprecipitated with STAT2 - specific antibody or vice versa . The amount of precipitated DNA was measured by Q - PCR . Bars represent mean values of three independent experiments ; error bars represent standard deviation ( SD ) .

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C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP-reChIP. BMDMs of wild type (WT) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with either STAT1-specific antibody and re-immunoprecipitated with STAT2-specific antibody or vice versa. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard deviation (SD).


D Impact of STAT1Y701F on delayed , STAT2 - mediated expression of IFN - induced genes . Stat1 - / - fibroblasts were transfected with plasmids driving expression of the indicated proteins . 24 h after transfection , 250 IU / ml of IFNβ was added to the transfected cells and ISG expression was determined by Q - PCR after 48h of cytokine treatment . Gene expression was measured by Q - PCR and normalized to Gapdh . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) and asterisks denote the level of statistical significance ( * , p ≤ 0 . 05 ) ; the p - values were calculated using paired ratio t - test .

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D Impact of STAT1Y701F on delayed, STAT2-mediated expression of IFN-induced genes. Stat1-/- fibroblasts were transfected with plasmids driving expression of the indicated proteins. 24 h after transfection, 250 IU/ml of IFNβ was added to the transfected cells and ISG expression was determined by Q-PCR after 48h of cytokine treatment. Gene expression was measured by Q-PCR and normalized to Gapdh. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM) and asterisks denote the level of statistical significance (*, p≤ 0.05); the p-values were calculated using paired ratio t-test.


A Analysis of STAT2 nuclear translocation by immunofluorescence . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice were seeded on cover slips and stimulated with 250 IU / mL of IFNβ for 30 min or 24 h . The cells were fixed and stained for STAT2 specific antibody followed by Alexafluor ® 488 conjugated secondary antibody ( green ) . Nuclei were stained with DAPI ( blue ) . Studies are representative of more than three independent experiments . The scale bars represent 10 µm . B Quantitative evaluation of STAT2nuclear translocation . The intensity of STAT2 - dependent immunofluorescence over DNA staining ( DAPI ) was quantified using ImageJ software in 20 cells from two independent experiments . Bars represent a mean with standard deviation ( SD ) and asterisks denote the level of statistical significance ( * * * , p ≤ 0 . 001 ) ; p - value was calculated using unpaired t - test .

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A Analysis of STAT2 nuclear translocation by immunofluorescence. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were seeded on cover slips and stimulated with 250 IU/mL of IFNβ for 30 min or 24 h. The cells were fixed and stained for STAT2 specific antibody followed by Alexafluor® 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Studies are representative of more than three independent experiments. The scale bars represent 10 µm.B Quantitative evaluation of STAT2nuclear translocation. The intensity of STAT2-dependent immunofluorescence over DNA staining (DAPI) was quantified using ImageJ software in 20 cells from two independent experiments. Bars represent a mean with standard deviation (SD) and asterisks denote the level of statistical significance (***, p≤ 0.001); p-value was calculated using unpaired t-test.


A Legionella pneumophila growth in unstimulated macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F , Stat1 - / - mice were seeded on in the 24 - well plates and infected with L . pneumophila ( JR32 Fla - , MOI 0 . 25 ) . The numbers of colony forming units ( CFUs ) were determined 24 h , 48 h and 72 h after infection on charcoal yeast extract plates ( CYE ) . The 0 time point was collected 1 . 5 h after the infection . B Legionella pneumophila growth in IFNβ - treated macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F , Stat1 - / - mice were seeded in 24 - well plates , treated with 500 U / mL of IFNβ and then infected with L . pneumophila ( JR32 Fla - , MOI 0 . 25 ) . The numbers of colony forming units ( CFUs ) were determined 24 h , 48 h and 72 h after infection on charcoal yeast extract plates ( CYE ) . The 0 time point was collected 1 . 5 h after the infection .

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A Legionella pneumophila growth in unstimulated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded on in the 24-well plates and infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.B Legionella pneumophila growth in IFNβ-treated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded in 24-well plates, treated with 500 U/mL of IFNβ and then infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.


A Western blot analysis of STAT1 tyrosine phosphorylation . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were infected with L . monocytogenes ( LO28 , MOI 10 ) for 5 or 6 h . Whole cell extracts were collected and tested in western blot for levels of total STAT1 and phosphorylation of STAT1 on tyrosine ( Y701 ) . The blots are representative of more than 3 independent experiments .

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A Western blot analysis of STAT1 tyrosine phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected with L. monocytogenes (LO28, MOI 10) for 5 or 6 h. Whole cell extracts were collected and tested in western blot for levels of total STAT1 and phosphorylation of STAT1 on tyrosine (Y701). The blots are representative of more than 3 independent experiments.


B Impact of Stat1Y701F mutation , or of deletion of ISGF3 subunits on the expression of the IFNβ gene . BMDMs of wild type ( WT ) , Stat1Y701F , Stat1 - / - , Stat2 - / - and IRF9 - / - mice were infected with L . monocytogenes ( LO28 , MOI 10 ) for 4 , 8 , 12 , 24 or 48 h . Levels of Ifnβ gene expression were determined by Q - PCR . Bars represent mean values of three independent experiments . Error bars represent standard error of mean ( SEM ) .

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B Impact of Stat1Y701F mutation, or of deletion of ISGF3 subunits on the expression of the IFNβ gene. BMDMs of wild type (WT), Stat1Y701F, Stat1-/-, Stat2-/- and IRF9-/- mice were infected with L. monocytogenes (LO28, MOI 10) for 4, 8, 12, 24 or 48 h. Levels of Ifnβ gene expression were determined by Q-PCR. Bars represent mean values of three independent experiments. Error bars represent standard error of mean (SEM).


A Impact of Stat1 deficiency or of STAT1Y701F mutation on the growth of Listeria monocytogenes in macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F and Stat1 - / - mice were infected with L . monocytogenes ( LO28 , MOI 10 ) . Colony forming unit ( CFU ) numbers were determined 1 , 2 , 4 , 6 or 8 h after infection by plating on brain - heart - infusion ( BHI ) agar plates . The graph represents biological triplicates and the data are represented as mean values . Error bars represent standard deviation ( SD ) and asterisks denote statistically significant differences ( ns , p > 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ) ; p - values were calculated using unpaired t - test .

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A Impact of Stat1 deficiency or of STAT1Y701F mutation on the growth of Listeria monocytogenes in macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F and Stat1-/- mice were infected with L. monocytogenes (LO28, MOI 10). Colony forming unit (CFU) numbers were determined 1, 2, 4, 6 or 8 h after infection by plating on brain-heart-infusion (BHI) agar plates. The graph represents biological triplicates and the data are represented as mean values. Error bars represent standard deviation (SD) and asterisks denote statistically significant differences (ns, p>0.05; **, p≤0.01; ***, p≤ 0.001); p-values were calculated using unpaired t-test.


B Survival of mice infected with L . monocytogenes . 10 wild type ( WT ) , Stat1Y701F and Stat1 - / - mice per group were infected by intraperitoneal injection of 1x 102 viable L . monocytogenes . Survival was monitored over 10 days . The study is representative of more than 3 independent experiments .

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B Survival of mice infected with L. monocytogenes. 10 wild type (WT), Stat1Y701F and Stat1-/- mice per group were infected by intraperitoneal injection of 1x 102 viable L. monocytogenes. Survival was monitored over 10 days. The study is representative of more than 3 independent experiments.


C Organ pathogen burdens of mice infected with L . monocytogenes . Wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were infected by intraperitoneal injection of 1x 102 viable L . monocytogenes . Number of colony forming units ( CFU ) in organs was determined at 48 h , 72 h or at the terminal stage of infection by plating homogenates on brain - heart - infusion ( BHI ) agar plates or Oxford agar plates ( for lungs ) . Dots represent pooled data of 3 independent experiments . Lines represent the median and asterisks denote statistically significant differences ( ns , p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ; * * * * , p ≤ 0 . 0001 ) ; p - values were calculated using Mann - Whitney test .

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C Organ pathogen burdens of mice infected with L. monocytogenes. Wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected by intraperitoneal injection of 1x 102 viable L. monocytogenes. Number of colony forming units (CFU) in organs was determined at 48 h, 72 h or at the terminal stage of infection by plating homogenates on brain-heart-infusion (BHI) agar plates or Oxford agar plates (for lungs). Dots represent pooled data of 3 independent experiments. Lines represent the median and asterisks denote statistically significant differences (ns, p>0.05; *, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001; ****, p≤ 0.0001); p-values were calculated using Mann-Whitney test.


A Immunohistochemical analysis of infection and inflammatory infiltrates . Wild type ( WT ) , Stat1Y701F and Stat1 - / - mice were infected by intraperitoneal injection of 1x102 viable L . monocytogenes for 48 h . Liver sections were examined by immunohistochemistry with L . monocytogenes or Ly6C / Ly6G specific antibody . The scale bars on 20x magnification images represent 100 µm and 50 µm on the 63x magnification images . B Quantitative evaluation of inflammatory infiltrates . Infiltrates representing the entire surface of sections from five animals per genotype were counted and categorized according to their size .

A Immunohistochemical analysis of infection and inflammatory infiltrates. Wild type (WT), Stat1Y701F and Stat1-/- mice were infected by intraperitoneal injection of 1x102 viable L. monocytogenes for 48 h. Liver sections were examined by immunohistochemistry with L. monocytogenes or Ly6C/Ly6G specific antibody. The scale bars on 20x magnification images represent 100 µm and 50 µm on the 63x magnification images.B Quantitative evaluation of inflammatory infiltrates. Infiltrates representing the entire surface of sections from five animals per genotype were counted and categorized according to their size.


C Liver pathology in infected mice . Wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were infected by intraperitoneal injection of 1x105 viable L . monocytogenes for 72 h . Serum was collected and tested for ALT activity . Lines represent the median and asterisks denote statistically significant differences ( * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ) ; p - values were calculated using unpaired t - test .

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C Liver pathology in infected mice. Wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected by intraperitoneal injection of 1x105 viable L. monocytogenes for 72 h. Serum was collected and tested for ALT activity. Lines represent the median and asterisks denote statistically significant differences (*, p≤ 0.05; ***, p≤ 0.001); p-values were calculated using unpaired t-test.


( B ) Downstream polarization of VE - PTP ( red ) in endothelial cells in the thoracic aorta . Blood flow : left to right . VE - cadherin , green . ( C ) Higher magnification views of a region similar to B , showing variability of VE - PTP polarization ranging from conspicuous ( upper ) to subtle ( lower ) . Nuclei , blue ( lamin A / C ) . (

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(B) Downstream polarization of VE-PTP (red) in endothelial cells in the thoracic aorta. Blood flow: left to right. VE-cadherin, green. (C) Higher magnification views of a region similar to B, showing variability of VE-PTP polarization ranging from conspicuous (upper) to subtle (lower). Nuclei, blue (lamin A/C). (


( D ) Dot plot of proportions of VE - PTP staining in upstream and downstream halves of endothelial cells in thoracic aorta of wild - type mouse . Each dot is one endothelial cell . Mean ± SEM , n = 15 cells .

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(D) Dot plot of proportions of VE-PTP staining in upstream and downstream halves of endothelial cells in thoracic aorta of wild-type mouse. Each dot is one endothelial cell. Mean ± SEM, n = 15 cells.


( C ) VE - PTP internalization in HUVEC into EEA1 endosomes ( arrowheads ) under static conditions and laminar flow of 15 dyn / cm2 of shear force for 5 , 10 , and 30 min . ( D ) Quantification of VE - PTP staining signals of images shown in C , which co - localize with EEA1 . Mean ± SEM , n = 10 pooled in 3 independent experiments (

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(C) VE-PTP internalization in HUVEC into EEA1 endosomes (arrowheads) under static conditions and laminar flow of 15 dyn/cm2 of shear force for 5, 10, and 30 min. (D) Quantification of VE-PTP staining signals of images shown in C, which co-localize with EEA1. Mean ± SEM, n= 10 pooled in 3 independent experiments (


( E ) HUVEC exposed to same conditions as in C , followed by fixation with ( upper row ) or without ( lower row ) permeabilization with 0 . 3 % Triton X - 100 , and staining for VE - PTP ( red ) and nuclei ( blue , Hoechst 33342 ) . ( F ) VE - PTP on cell surface measured by cell - based ELISA assay of HUVEC after exposure as in C and D . Red and black lines show measurements with anti - VE - PTP antibody and isotype control antibody . Mean ± SEM , n = 6 independent experiments / group .

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(E) HUVEC exposed to same conditions as in C, followed by fixation with (upper row) or without (lower row) permeabilization with 0.3% Triton X-100, and staining for VE-PTP (red) and nuclei (blue, Hoechst 33342). (F) VE-PTP on cell surface measured by cell-based ELISA assay of HUVEC after exposure as in C and D. Red and black lines show measurements with anti-VE-PTP antibody and isotype control antibody. Mean ± SEM, n = 6 independent experiments/group. D


( C - D ) Strong Tie2 - pY992 ( green ) staining at cell borders and VE - PTP ( red ) downstream polarization in outer curvature in C in contrast to weak , dispersed staining for Tie2 - pY992 and VE - PTP in inner curvature in D of aortic arch of wild - type mouse .

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(C-D) Strong Tie2-pY992 (green) staining at cell borders and VE-PTP (red) downstream polarization in outer curvature in C in contrast to weak, dispersed staining for Tie2-pY992 and VE-PTP in inner curvature in D of aortic arch of wild-type mouse.


( E - F ) Tie2 - pY992 ( green ) at endothelial junctions marked by VE - cadherin ( red ) . ( E ) Contrasting patterns and intensity of Tie2 - pY992 in outer and inner curvatures . ( F ) Tie2 - pY992 mean fluorescence intensity at junctions ( normalized to outer curvature = 1 . 0 ) . Mean ± SEM , n = 6 mice / group .

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(E-F) Tie2-pY992 (green) at endothelial junctions marked by VE-cadherin (red). (E) Contrasting patterns and intensity of Tie2-pY992 in outer and inner curvatures. (F) Tie2-pY992 mean fluorescence intensity at junctions (normalized to outer curvature = 1.0). Mean ± SEM, n = 6 mice/group.


( E ) Comparison of distribution of fluorescence intensities of extravasated antibody showing significantly stronger fluorescence due to greater leakage in inner curvature . ( F ) Mean fluorescence intensity value for each mouse in the two groups shown in E . Mean ± SEM , n = 7 mice / group . (

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(E) Comparison of distribution of fluorescence intensities of extravasated antibody showing significantly stronger fluorescence due to greater leakage in inner curvature. (F) Mean fluorescence intensity value for each mouse in the two groups shown in E. Mean ± SEM, n = 7 mice/group. (


( G ) Localized distribution of intense leakage ( red ) around ostium of an intercostal artery ( arrow ) . VE - cadherin ( green ) . ( H ) Opposite distributions of extravasation and VE - PTP polarization in endothelial cells in thoracic aorta . Left and right panels are the same image with colors switched to highlight leakage ( red ) and polarized VE - PTP ( green , arrowheads ) on the left , and polarized VE - PTP ( red , arrowheads ) , VE - cadherin ( green ) , and leakage ( blue ) on the right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Localized distribution of intense leakage (red) around ostium of an intercostal artery (arrow). VE-cadherin (green). (H) Opposite distributions of extravasation and VE-PTP polarization in endothelial cells in thoracic aorta. Left and right panels are the same image with colors switched to highlight leakage (red) and polarized VE-PTP (green, arrowheads) on the left, and polarized VE-PTP (red, arrowheads), VE-cadherin (green), and leakage (blue) on the right. D


( B ) Measurements of Tie2 - pY992 staining intensity at intercellular junctions in aortic arch endothelial cells showing lower values in the inner curvature than in the upper curvature of VE - PTPfl / fl mice but equally high values in both regions of VE - PTPiECKO mice . Normalized to mean value for outer curvature of VE - PTPfl / fl mice scaled to 1 . 0 . Mean ± SEM , n = 6 mice / group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Measurements of Tie2-pY992 staining intensity at intercellular junctions in aortic arch endothelial cells showing lower values in the inner curvature than in the upper curvature of VE-PTPfl/fl mice but equally high values in both regions of VE-PTPiECKO mice. Normalized to mean value for outer curvature of VE-PTPfl/fl mice scaled to 1.0. Mean ± SEM, n = 6 mice/group.


( C ) HUVEC transfected with control siRNA or VE - PTP siRNA and exposed to static or laminar shear force at 15 dyn / cm2 for 30 min , followed by immunoprecipitation of Tie2 . Left . Blot showing immunoprecipitates ( top ) or cell lysates ( bottom ) immunoblotted for pan - phosphotyrosine ( antibody 4G10 ) , Tie2 , or VE - PTP . Ratio of staining for phosphorylated Tie2 and total Tie2 ( Tie2 - pY to Tie2 , n = 4 experiments ) . Right . Relative Tie2 - pY levels in HUVEC after control siRNA or VE - PTP siRNA . Normalized to static control = 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HUVEC transfected with control siRNA or VE-PTP siRNA and exposed to static or laminar shear force at 15 dyn/cm2 for 30 min, followed by immunoprecipitation of Tie2. Left. Blot showing immunoprecipitates (top) or cell lysates (bottom) immunoblotted for pan-phosphotyrosine (antibody 4G10), Tie2, or VE-PTP. Ratio of staining for phosphorylated Tie2 and total Tie2 (Tie2-pY to Tie2, n = 4 experiments). Right. Relative Tie2-pY levels in HUVEC after control siRNA or VE-PTP siRNA. Normalized to static control = 1.


( D ) Effect of shear stress on the phosphorylation levels of Tie2 Y992 at cellular junctions . HUVECs treated with either control siRNA ( left ) or Tie2 siRNA ( right ) , were exposed to 15 dyn / cm2 shear stress for 5 min . The resulting cells were fixed and stained for Tie2 - pY992 ( red ) and VE - cadherin ( green ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of shear stress on the phosphorylation levels of Tie2 Y992 at cellular junctions. HUVECs treated with either control siRNA (left) or Tie2 siRNA (right), were exposed to 15 dyn/cm2 shear stress for 5 min. The resulting cells were fixed and stained for Tie2-pY992 (red) and VE-cadherin (green).


( E ) HUVECs cultured on biotinylated gelatin were treated either with control siRNA , VE - PTP siRNA , Tie2 siRNA or VE - cadherin siRNA , then exposed to 15 dyn / cm2 shear stress for 30 minutes followed by 3 min incubation with Streptavidin - Alexa647 ( red ) , washing , fixation and staining for VE - cadherin ( green ) . ( F and G ) Quantification of Streptavidin leakage area per image . Mean ± SEM , n = 19 pooled in ten independent experiments ( F ) , and n = 6 pooled in three independent experiments ( G ) .

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

(E) HUVECs cultured on biotinylated gelatin were treated either with control siRNA, VE-PTP siRNA, Tie2 siRNA or VE-cadherin siRNA, then exposed to 15 dyn/cm2 shear stress for 30 minutes followed by 3 min incubation with Streptavidin-Alexa647 (red), washing, fixation and staining for VE-cadherin (green). (F and G) Quantification of Streptavidin leakage area per image. Mean ± SEM, n = 19 pooled in ten independent experiments (F), and n = 6 pooled in three independent experiments (G). D


( B ) Fractional area occupied by Oil Red O - stained atheromas in aortic arch , brachiocephalic artery , at intercostal artery ostia , and away from ostia . Each dot is value for one mouse . Mean ± SEM , n = 13 - 18 mice / group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fractional area occupied by Oil Red O-stained atheromas in aortic arch, brachiocephalic artery, at intercostal artery ostia, and away from ostia. Each dot is value for one mouse. Mean ± SEM, n = 13-18 mice/group.


( D - E ) Fluorescence intensity distributions in D and mean intensities in E of extravasated anti - fibrinogen antibody in inner curvature of ApoE - / - / VE - PTPfl / fl mice and ApoE - / - / VE - PTPiECKO mice on high - fat diet for 7 wk . Mean ± SEM , n = 7 mice / group .

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

(D-E) Fluorescence intensity distributions in D and mean intensities in E of extravasated anti-fibrinogen antibody in inner curvature of ApoE-/-/VE-PTPfl/fl mice and ApoE-/-/VE-PTPiECKO mice on high-fat diet for 7 wk. Mean ± SEM, n = 7 mice/group.


( C ) Measurements of Tie2 - pY992 intensity at intercellular junctions in aortic arch of mice shown in B . Mean ± SEM , n = 8 mice / group .

O O O O O O O O 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) Measurements of Tie2-pY992 intensity at intercellular junctions in aortic arch of mice shown in B. Mean ± SEM, n = 8 mice/group.


( E ) Fractional area occupied by Oil Red O - stained atheromas in aortic arch , brachiocephalic artery , at intercostal artery ostia , and away from ostia . Each dot is value for one mouse . Mean ± SEM , n = 17 mice / group .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fractional area occupied by Oil Red O-stained atheromas in aortic arch, brachiocephalic artery, at intercostal artery ostia, and away from ostia. Each dot is value for one mouse. Mean ± SEM, n = 17 mice/group.


B . Coronal section of a P3 spheroid embedded with paraffin and stained with DAPI ( blue ) , LDHA ( green ) , and LDHB ( red ) . Magnification boxes show different areas as depicted in the main image . Quantification of LDHA and LDHB staining was performed on the spheroid areas as indicated in the graphs . Scale bar : 100 µm . Data are represented as mean ± s . d . ( 5 - 8 fields per area of interest , one representative spheroid of 3 independent invading spheroids )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Coronal section of a P3 spheroid embedded with paraffin and stained with DAPI (blue), LDHA (green), and LDHB (red). Magnification boxes show different areas as depicted in the main image. Quantification of LDHA and LDHB staining was performed on the spheroid areas as indicated in the graphs. Scale bar: 100 µm. Data are represented as mean ± s.d. (5-8 fields per area of interest, one representative spheroid of 3 independent invading spheroids)


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

A. Western blot analysis of LDHA and LDHB from P3 cells upon exposure to 21% or 0.1% O2 for 6, 24, 48, and 72 h. The graphs represent densitometry quantification of immunoblots normalized to tubulin (n = 4 independent experiments). Data are represented as mean ± s.d. and analyzed using One-Way ANOVA following Dunnett's multiple comparisons tests. LDHA: O2 21% vs O2 0.1% 6h, P>0.99; O2 21% vs O2 0.1% 24h, P=0.15; O2 21% vs O2 0.1% 48h, P=0.0009; O2 21% vs O2 0.1% 72h, P<0.0001. LDHB: O2 21% vs O2 0.1% 6h, P>0.99; O2 21% vs O2 0.1% 24h, P>0.99; O2 21% vs O2 0.1% 48h, P=0.97; O2 21% vs O2 0.1% 72h, P=0.88.


A . Western blot analysis of LDHA and LDHB expression in P3 cells transduced with CRISPR - Cas9 lentiviral vectors with scramble sequence ( sgControl ) or against LDHA ( sgLDHA ) , LDHB ( sgLDHB ) or both ( sgLDHA / B ) . Knockout ( KO ) cells were exposed to 21 % or 1 % O2 for 48 h .

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

A. Western blot analysis of LDHA and LDHB expression in P3 cells transduced with CRISPR-Cas9 lentiviral vectors with scramble sequence (sgControl) or against LDHA (sgLDHA), LDHB (sgLDHB) or both (sgLDHA/B). Knockout (KO) cells were exposed to 21% or 1% O2 for 48 h.


D . Left : P3 spheroid growth recorded over one week at 1 % O2 ( n = 3 independent experiments , one experiment including 16 spheroids per condition ) . Data are represented as mean and analyzed using Kruskal - Wallis test followed by Dunn ' s multiple comparisons test . Spheroid growth : sgCont vs sgLDHA , P = 0 . 0003 ; sgCont vs sgLDHB , P = 0 . 78 ; sgCont vs sgLDHA / B , P < 0 . 0001 . Dead area : sgCont vs sgLDHA , P = 0 . 19 ; sgCont vs sgLDHB , P < 0 . 0001 ; sgCont vs sgLDHA / B , P < 0 . 0001 . Right : Viability of spheroids at one week , incubated with calcein ( green ) or ethidium homodimer - 1 ( red ) . Scale bar : 200 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Left: P3 spheroid growth recorded over one week at 1% O2 (n = 3 independent experiments, one experiment including 16 spheroids per condition). Data are represented as mean and analyzed using Kruskal-Wallis test followed by Dunn's multiple comparisons test. Spheroid growth: sgCont vs sgLDHA, P=0.0003; sgCont vs sgLDHB, P=0.78; sgCont vs sgLDHA/B, P<0.0001. Dead area: sgCont vs sgLDHA, P=0.19; sgCont vs sgLDHB, P<0.0001; sgCont vs sgLDHA/B, P<0.0001. Right: Viability of spheroids at one week, incubated with calcein (green) or ethidium homodimer-1 (red). Scale bar: 200 µm.


E . P3 spheroid invasion in collagen I gel , incubated 24 h at 21 % or 0 . 1 % O2 . Invasion rate is expressed as fold change from controls ( n = 4 independent experiments , one experiment including 6 - 8 spheroids per condition ) . Data are represented as mean and analyzed using Two - way ANOVA following by Tukey ' s multiple comparisons test : sgCont 21 % vs sgLDHA 21 % , P = 0 . 003 ; sgCont 0 . 1 % vs sgLDHA 0 . 1 % , P < 0 . 0001 ; sgCont 21 % vs sgLDHB 21 % , P < 0 . 0001 ; sgCont 0 . 1 % vs sgLDHB 0 . 1 % , P < 0 . 0001 ; sgCont 21 % vs sgLDHA / B 21 % , P < 0 . 0001 ; sgCont 0 . 1 % vs sgLDHA / B 0 . 1 % , P < 0 . 0001 . Images of representative of control or LDHA / B KO spheroids . Scale bar : 100 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. P3 spheroid invasion in collagen I gel, incubated 24 h at 21% or 0.1% O2. Invasion rate is expressed as fold change from controls (n = 4 independent experiments, one experiment including 6-8 spheroids per condition). Data are represented as mean and analyzed using Two-way ANOVA following by Tukey's multiple comparisons test: sgCont 21% vs sgLDHA 21%, P=0.003; sgCont 0.1% vs sgLDHA 0.1%, P<0.0001; sgCont 21% vs sgLDHB 21%, P<0.0001; sgCont 0.1% vs sgLDHB 0.1%, P<0.0001; sgCont 21% vs sgLDHA/B 21%, P<0.0001; sgCont 0.1% vs sgLDHA/B 0.1%, P<0.0001. Images of representative of control or LDHA/B KO spheroids. Scale bar: 100 µm.


F - G . A first cohort of mice was sacrificed when one mouse reached a limit point , brains was extract , snap frozen in liquid nitrogen , sliced and stained . For the second cohort , each mouse was sacrificed when it reached a limit point allowing the draw of survival curves . Left : Immunofluorescence staining of Nestin ( top and middle ) and CD31 ( bottom ) of control and LDHA / B KO P3 tumor section . Scale bars : 2 mm ( top ) , 250 µm ( middle ) and 100 µm ( bottom ) . Right : Graphs represent tumor core and invasion area of control and LDHA / B KO P3 tumors in mm2 ( n = 5 mice per group , average of 5 - 6 sections per tumor ) . Data are represented as mean . For F , data are analyzed using One - way ANOVA following by Dunnett ' s multiple comparisons test . Core : sgCont vs sgLDHA , P = 0 . 48 ; sgCont vs sgLDHB , P = 0 . 99 . Invasion : sgCont vs sgLDHA , P = 0 . 40 ; sgCont vs sgLDHB , P = 0 . 81 . For G , data are analyzed using unpaired t test . Core : sgCont vs sgLDHA / B , P = 0 . 005 . Invasion : sgCont vs sgLDHA / B , P = 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A first cohort of mice was sacrificed when one mouse reached a limit point, brains was extract, snap frozen in liquid nitrogen, sliced and stained. For the second cohort, each mouse was sacrificed when it reached a limit point allowing the draw of survival curves. Left: Immunofluorescence staining of Nestin (top and middle) and CD31 (bottom) of control and LDHA/B KO P3 tumor section. Scale bars: 2 mm (top), 250 µm (middle) and 100 µm (bottom). Right: Graphs represent tumor core and invasion area of control and LDHA/B KO P3 tumors in mm2 (n = 5 mice per group, average of 5-6 sections per tumor). Data are represented as mean. For F, data are analyzed using One-way ANOVA following by Dunnett's multiple comparisons test. Core: sgCont vs sgLDHA, P=0.48; sgCont vs sgLDHB, P=0.99. Invasion: sgCont vs sgLDHA, P=0.40; sgCont vs sgLDHB, P=0.81. For G, data are analyzed using unpaired t test. Core: sgCont vs sgLDHA/B, P=0.005. Invasion: sgCont vs sgLDHA/B, P=0.01.


A . Western blot analysis of mitochondrial respiratory chain subunits from LDH KO P3 cells upon exposure to 21 % or 0 . 1 % O2 . Roman numbers indicate the respiratory complex number . The diagram below represents densitometry quantification of the immunoblots normalized to vinculin and expressed as log2 foldchange to control cells in 21 % and 0 . 1 % O2 ( n = 3 independent experiments ) .

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

A. Western blot analysis of mitochondrial respiratory chain subunits from LDH KO P3 cells upon exposure to 21% or 0.1% O2. Roman numbers indicate the respiratory complex number. The diagram below represents densitometry quantification of the immunoblots normalized to vinculin and expressed as log2 foldchange to control cells in 21% and 0.1% O2 (n = 3 independent experiments).


B . Epifluorescence ( top ) and quantitative image analysis ( bottom ) of immune stained mitochondria ( TOM20 , green ) from LDH KO P3 cells ( Phalloidin , red ; DAPI , blue ) upon exposure to 0 . 1 % O2 ( n = 2 independent experiments , 28 - 32 cells per group ) . Data are represented as mean and analyzed using One - way ANOVA following by Tukey ' s multiple comparisons test . Mitochondrial mass : sgCont vs sgLDHA / B , P = 0 . 005 ; sgLDHA / sgLDHB vs sgLDHA / B , p = 0 . 006 . Network aspect ratio : sgCont vs sgLDHA / B , P = 0 . 03 ; sgLDHA vs sgLDHA / B , P = 0 . 02 ; sgLDHB vs sgLDHA / B , p = 0 . 04 . Mitochondrial mass corresponds to the area covered by the mitochondria relative to the entire cell area . Network aspect ratio is the ratio of the major axis to minor axis of the mitochondrial network . Scale bar : 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Epifluorescence (top) and quantitative image analysis (bottom) of immune stained mitochondria (TOM20, green) from LDH KO P3 cells (Phalloidin, red; DAPI, blue) upon exposure to 0.1% O2 (n = 2 independent experiments, 28-32 cells per group). Data are represented as mean and analyzed using One-way ANOVA following by Tukey's multiple comparisons test. Mitochondrial mass: sgCont vs sgLDHA/B, P=0.005; sgLDHA/sgLDHB vs sgLDHA/B, p=0.006. Network aspect ratio: sgCont vs sgLDHA/B, P=0.03; sgLDHA vs sgLDHA/B, P=0.02; sgLDHB vs sgLDHA/B, p=0.04. Mitochondrial mass corresponds to the area covered by the mitochondria relative to the entire cell area. Network aspect ratio is the ratio of the major axis to minor axis of the mitochondrial network. Scale bar: 10 µm.


E . Kaplan - Meier survival curves of xenotransplanted mice with LDHA / B KO ( red ) or control ( blue ) P3 cells , treated with phenformin or irradiated with 10 Gy ( n = 6 - 8 mice per group ) . Data are analyzed using Log - rank ( Mantel - Cox ) test : sgCont Vehicle vs sgCont Phenformin , P = 0 . 019 ; sgCont Vehicle vs sgCont Irradiation , P = 0 . 0002 ; sgCont Irradiation vs sgLDHA / B Irradiation , P = 0 . 024 .

O O O O 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 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

E. Kaplan-Meier survival curves of xenotransplanted mice with LDHA/B KO (red) or control (blue) P3 cells, treated with phenformin or irradiated with 10 Gy (n = 6-8 mice per group). Data are analyzed using Log-rank (Mantel-Cox) test: sgCont Vehicle vs sgCont Phenformin, P=0.019; sgCont Vehicle vs sgCont Irradiation, P=0.0002; sgCont Irradiation vs sgLDHA/B Irradiation, P=0.024.


D . Left : P3 spheroid growth recorded over 72 h during incubation with stiripentol at 0 . 1 % O2 ( n = 3 independent experiments , one experiment including 8 spheroids per condition ) . Data are represented as mean ± s . d . and growth at 72 h are analyzed using unpaired t test : P < 0 . 0001 . Right : Viability of spheroids at 72 h , incubated with calcein ( green ) or ethidium homodimer - 1 ( red ) . Scale bar : 100 µm . E . Left : P3 spheroid invasion in collagen I gel , incubated 24 h with 500 µM stiripentol at 0 . 1 % O2 . Invasion rate is expressed as fold change from control ( n = 3 independent experiments , one experiment including 8 spheroids per condition ) . Data are represented as mean and analyzed using unpaired t test : P < 0 . 0001 . Right : Representative images of invasive spheroids for each condition . Scale bar : 100 µm .

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

D. Left: P3 spheroid growth recorded over 72 h during incubation with stiripentol at 0.1% O2 (n = 3 independent experiments, one experiment including 8 spheroids per condition). Data are represented as mean ± s.d. and growth at 72 h are analyzed using unpaired t test: P<0.0001. Right: Viability of spheroids at 72 h, incubated with calcein (green) or ethidium homodimer-1 (red). Scale bar: 100 µm. E. Left: P3 spheroid invasion in collagen I gel, incubated 24 h with 500 µM stiripentol at 0.1% O2. Invasion rate is expressed as fold change from control (n = 3 independent experiments, one experiment including 8 spheroids per condition). Data are represented as mean and analyzed using unpaired t test: P<0.0001. Right: Representative images of invasive spheroids for each condition. Scale bar: 100 µm.


F . Kaplan - Meier survival curves of xenotransplanted mice with P3 cells . Mice were treated either with vehicle ( blue ) or stiripentol ( red ) ( n = 15 - 16 mice per group ) . Data are analyzed using Log - rank ( Mantel - Cox ) test : P = 0 .

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

F. Kaplan-Meier survival curves of xenotransplanted mice with P3 cells. Mice were treated either with vehicle (blue) or stiripentol (red) (n = 15-16 mice per group). Data are analyzed using Log-rank (Mantel-Cox) test: P=0.002


A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( A ) left untreated or treated with increasing concentrations of LLO for 15 min . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) .

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

A-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (A) left untreated or treated with increasing concentrations of LLO for 15 min. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).


( A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( B ) left uninfected ( U ) or infected with wt or Δhly Lm for 1 h . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) .

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

(A-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (B) left uninfected (U) or infected with wt or Δhly Lm for 1 h. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).


( A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( C ) infected with wt Lm for the indicated time points . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) .

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

(A-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (C) infected with wt Lm for the indicated time points. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).


( D ) Confocal microscopy images of HeLa cells : ( D ) left untreated or treated with LLO ( 0 . 5 nM , 15 min ) , immunolabelled for NMHCIIA ( green ) , Gp96 ( red ) and stained with DAPI ( blue ) . Arrows point to various NMHCIIA - Gp96 positive cortical bundles in different cell . Insets show high magnification image ( E ) Quantification of Pearson ' s correlation coefficient ( PCC ) within : uninfected cells ( U ) , LLO - treated cells , cortical NMHCIIA bundles or equivalent size control cellular ROI . ( Ctrl ROI ) Data are mean ± SEM ( n = 6 ) ; p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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

(D) Confocal microscopy images of HeLa cells: (D) left untreated or treated with LLO (0.5 nM, 15 min), immunolabelled for NMHCIIA (green), Gp96 (red) and stained with DAPI (blue). Arrows point to various NMHCIIA-Gp96 positive cortical bundles in different cell. Insets show high magnification image (E) Quantification of Pearson's correlation coefficient (PCC) within: uninfected cells (U), LLO-treated cells, cortical NMHCIIA bundles or equivalent size control cellular ROI. (Ctrl ROI) Data are mean±SEM (n=6); p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.05, **p<0.01, ***p<0.001


( F ) Confocal microscopy images of HeLa cells infected with wt or Δhly Lm for 6 h immunolabelled for NMHCIIA ( green ) , Gp96 ( red ) Lm ( blue ) and stained with DAPI ( white ) . Arrows indicate NMHCIIA - Gp96 cortical bundles at cortical sites close to wt Lm . ( D and F ) Scale bar - 10 μm . See also Figs EV1 and EV2 .

(F) Confocal microscopy images of HeLa cells infected with wt or Δhly Lm for 6 h immunolabelled for NMHCIIA (green), Gp96 (red) Lm (blue) and stained with DAPI (white). Arrows indicate NMHCIIA-Gp96 cortical bundles at cortical sites close to wt Lm. (D and F) Scale bar - 10 μm. See also Figs EV1 and EV2.


( A - B ) Sequential frames of time - lapse confocal microscopy sequence of LLO - treated HeLa cells expressing ( A ) GFP - NMHCIIA . LLO was added to culture medium 10 seconds before t0 . DIC - differential interference contrast . ( A ) Arrows indicate NMHCIIA bundles at PM blebbing sites .

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

(A-B) Sequential frames of time-lapse confocal microscopy sequence of LLO-treated HeLa cells expressing (A) GFP-NMHCIIA . LLO was added to culture medium 10 seconds before t0. DIC - differential interference contrast. (A) Arrows indicate NMHCIIA bundles at PM blebbing sites.


( A - B ) Sequential frames of time - lapse confocal microscopy sequence of LLO - treated HeLa cells expressing ( B ) simultaneously GFP - NMHCIIA and mcherry - KDEL . LLO was added to culture medium 10 seconds before t0 . DIC - differential interference contrast . ( B ) Highlights depicting ER structures within NMHCIIA bundles and PM blebs . Arrows indicate cortical ER surrounding NMHCIIA accumulations , arrowheads indicate contact points between ER vesicles and NMHCIIA cables and asterisks point to ER vacuoles within PM

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Sequential frames of time-lapse confocal microscopy sequence of LLO-treated HeLa cells expressing (B) simultaneously GFP-NMHCIIA and mcherry-KDEL. LLO was added to culture medium 10 seconds before t0. DIC - differential interference contrast. (B) Highlights depicting ER structures within NMHCIIA bundles and PM blebs. Arrows indicate cortical ER surrounding NMHCIIA accumulations, arrowheads indicate contact points between ER vesicles and NMHCIIA cables and asterisks point to ER vacuoles within PM blebs


( C ) Confocal microscopy images of HeLa cells left untreated ( Control ) or treated with LLO ( 0 . 5 nM 15 min ) . Cells were stained with FITC - WGA ( Plasma membrane , PM - red ) and immunolabelled for NMHCIIA ( green ) . Insets show PM blebs and arrows indicate recruitment of NMHCIIA bundles to PM blebs associated ( 1 ) or detached ( 2 ) from the cell body .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of HeLa cells left untreated (Control) or treated with LLO (0.5 nM 15 min). Cells were stained with FITC-WGA (Plasma membrane, PM-red) and immunolabelled for NMHCIIA (green). Insets show PM blebs and arrows indicate recruitment of NMHCIIA bundles to PM blebs associated (1) or detached (2) from the cell body.


( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( D ) LLO pre - incubated with cholesterol ( LLOCHT )

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

(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (D) LLO pre-incubated with cholesterol (LLOCHT)


( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( E ) LLO in medium supplemented with 140 mM K + ( HighK + ) and LLO in Ca2 + - free medium Ca2 + ( Ca2 + free )

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

(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (E) LLO in medium supplemented with 140 mM K+(HighK+) and LLO in Ca2+-free medium Ca2+ (Ca2+free)


( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( F ) LLO in the presence of 25 μM blebbistatin ( BB ) .

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

(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (F) LLO in the presence of 25 μM blebbistatin (BB).


( G ) Confocal microscopy images of HeLa cells treated as indicated and immunolabelled for ER - KDEL ( blue ) , NMHCIIA ( green ) and Gp96 ( red ) .

O O O O O O O O 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) Confocal microscopy images of HeLa cells treated as indicated and immunolabelled for ER-KDEL (blue), NMHCIIA (green) and Gp96 (red).


( H ) Confocal microscopy images of HeLa cells treated with aerolysin and immunolabelled for NMHCIIA ( green ) and Gp96 ( red ) . Insets show NMHCIIA bundles and arrows indicate association between Gp96 and NMHCIIA .

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

(H) Confocal microscopy images of HeLa cells treated with aerolysin and immunolabelled for NMHCIIA (green) and Gp96 (red). Insets show NMHCIIA bundles and arrows indicate association between Gp96 and NMHCIIA.


( I ) Immunoblots of NMHCIIA and Gp96 levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with LLO ( 0 . 5 nM , 15 min ) , SLO ( 1 . 5 μg / ml , 30 min ) ( SLO ) or aerolysin ( 0 . 2 nM , 40 min ) ( AL ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots of NMHCIIA and Gp96 levels from Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min), SLO (1.5 μg/ml, 30 min) (SLO) or aerolysin (0.2 nM, 40 min) (AL).


( A ) Confocal microscopy images of shCtrl , shGp96 or shNMHCIIA HeLa cells treated with LLO ( 0 . 5 nM , 15 min ) and immunolabelled for KDEL - proteins ( blue ) , NMHCIIA ( green ) and Gp96 ( red ) . Insets show compact NMHCIIA bundles in shCtrl cells and dispersed NMHCIIA bundles in shGp96 cells . Scale bar - 10 μm . ( B - D ) Quantification of the % of cells harbouring NMHCIIA bundles after incubation with ( B ) LLO ( 0 . 5 nM , 15 min ) , ( C ) aerolysin ( 0 . 2 nM , 40 min ) or ( D ) with wt Lm . Values are the mean ± SEM ( n ≥ 3 ) , p - values were calculated using ( B - C ) one - way - ANOVA with Dunnett post hoc analyses and ( D ) two - tailed un - paired Student ' s t - test , * p < 0 . 5 , * * * p < 0 . 001 . For shNMHCIIA , bundles were detected following actin staining ( Fig EV3C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of shCtrl, shGp96 or shNMHCIIA HeLa cells treated with LLO (0.5 nM, 15 min) and immunolabelled for KDEL-proteins (blue), NMHCIIA (green) and Gp96 (red). Insets show compact NMHCIIA bundles in shCtrl cells and dispersed NMHCIIA bundles in shGp96 cells. Scale bar - 10 μm. (B-D) Quantification of the % of cells harbouring NMHCIIA bundles after incubation with (B) LLO (0.5 nM, 15 min), (C) aerolysin (0.2 nM, 40 min) or (D) with wt Lm. Values are the mean ±SEM (n≥3), p-values were calculated using (B-C) one-way-ANOVA with Dunnett post hoc analyses and (D) two-tailed un-paired Student's t-test, *p<0.5, ***p<0.001. For shNMHCIIA, bundles were detected following actin staining (Fig EV3C).


( E - F ) Number of ( E ) blebs per cell or ( F ) retracting blebs per cell evaluated by time - lapse microscopy analysis of LLO - treated shCtrl or shGp96 cells . shCtrl n = 32 cells and shGp96 n = 40 cells , p - values were calculated using two - tailed un - paired Student ' s t - test * p < 0 . 5 . ( G ) Sequential frames of time - lapse microscopy analysis of LLO - treated HeLa cells expressing GFP - NMHCIIA ( shCtrl , shGp96 and shCtrl with 25 μM Blebbistatin - shCtrl - BB ) . LLO was added to culture medium 10 seconds before t0 .

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

(E-F) Number of (E) blebs per cell or (F) retracting blebs per cell evaluated by time-lapse microscopy analysis of LLO-treated shCtrl or shGp96 cells. shCtrl n=32 cells and shGp96 n=40 cells, p-values were calculated using two-tailed un-paired Student's t-test *p<0.5. (G) Sequential frames of time-lapse microscopy analysis of LLO-treated HeLa cells expressing GFP-NMHCIIA (shCtrl, shGp96 and shCtrl with 25 μM Blebbistatin - shCtrl-BB). LLO was added to culture medium 10 seconds before t0.


( H ) Immunoblots of Ser19 - phosphorylated MRLC ( pMRLC ) , MRLC and actin levels from shCtrl or shGp96 cells left untreated ( U ) or treated with 0 . 1 nM LLO for the indicated time points . Quantification of pMRLC levels corresponds to the mean ± SEM ( n ≥ 3 ) , p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 , * * * p < 0 . 001 . See also Appendix Fig S2 and Fig EV3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots of Ser19-phosphorylated MRLC (pMRLC), MRLC and actin levels from shCtrl or shGp96 cells left untreated (U) or treated with 0.1 nM LLO for the indicated time points. Quantification of pMRLC levels corresponds to the mean ± SEM (n≥3), p-values were calculated using one-way-ANOVA with Tukey post hoc analyses **p<0.01, ***p<0.001. See also Appendix Fig S2 and Fig EV3.


( Ai ) Confocal microscopy images of LLO - treated HeLa cells immunolabelled for the indicated proteins . Insets show colocalization between NMHCIIA and WRAMP components at cortical bundles . ( Aii ) Quantification of Pearson ' s correlation coefficient ( PCC ) between NMHCIIA and the indicated proteins within entire LLO - treated cells , cortical bundles or equivalent size control cellular ROI ( Ctrl ROI ) . For Calpain 2 cortical bundles were defined by actin staining . Data are representative of one experiment repeated 3 independent times with similar results . p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 ; * * * p < 0 . 001 , ns - non

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

(Ai) Confocal microscopy images of LLO-treated HeLa cells immunolabelled for the indicated proteins. Insets show colocalization between NMHCIIA and WRAMP components at cortical bundles. (Aii) Quantification of Pearson's correlation coefficient (PCC) between NMHCIIA and the indicated proteins within entire LLO-treated cells, cortical bundles or equivalent size control cellular ROI (Ctrl ROI). For Calpain 2 cortical bundles were defined by actin staining. Data are representative of one experiment repeated 3 independent times with similar results. p-values were calculated using one-way-ANOVA with Tukey post hoc analyses ** p<0.01; *** p<0.001, ns - non significant


( B ) Immunoblots of NMHCIIA , Gp96 and Filamin A levels from WCL and Gp96 IP of HeLa cells left untreated ( U ) or treated with LLO ( 0 . 5 nM , 15 min ) ( LLO ) .

O O O O O O O O O O O O O O O O O O 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) Immunoblots of NMHCIIA, Gp96 and Filamin A levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min) (LLO).


( C ) Immunoblots of NMHCIIA and Gp96 levels from WCL and Gp96 IP of HeLa cells left untreated ( U ) or treated with 25 μg / ml of Wnt5a for the indicated time .

O O O O O O O O O O O O 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) Immunoblots of NMHCIIA and Gp96 levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with 25 μg/ml of Wnt5a for the indicated time.


( D ) Confocal microscopy images of HeLa cells untreated ( Mock ) or treated with 25 μg / ml of Wnt5a for 30 min . Cells were immunolabelled for ER - KDEL ( blue ) , Gp96 ( red ) and NMHCIIA ( green ) . Arrows indicate polarised localization of NMHCIIA , ER - KDEL and Gp96 in Wnt5a - treated cells .

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

(D) Confocal microscopy images of HeLa cells untreated (Mock) or treated with 25 μg/ml of Wnt5a for 30 min. Cells were immunolabelled for ER-KDEL (blue), Gp96 (red) and NMHCIIA (green). Arrows indicate polarised localization of NMHCIIA, ER-KDEL and Gp96 in Wnt5a-treated cells.


( E - F ) Confocal microscopy images of shCtlr , shGp96 or shNMHCIIA HeLa cells stained for actin ( red ) and DAPI ( blue ) and immunolabelled for ( E ) NMHCIIA ( green ) or ( F ) focal adhesion kinase ( FAK ) ( green ) . ( A , D - F ) Scale bar - 10 μm . Insets show sites with focal adhesion points and arrows indicate stress fibers or distinct focal adhesion points . Quantification of the % of cells with ( G ) stress fibers or ( H ) focal adhesion points labelled by FAK .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Confocal microscopy images of shCtlr, shGp96 or shNMHCIIA HeLa cells stained for actin (red) and DAPI (blue) and immunolabelled for (E) NMHCIIA (green) or (F) focal adhesion kinase (FAK) (green). (A, D-F) Scale bar - 10 μm. Insets show sites with focal adhesion points and arrows indicate stress fibers or distinct focal adhesion points. Quantification of the % of cells with (G) stress fibers or (H) focal adhesion points labelled by FAK.


( I - J ) Wound closure assay . ( I ) Sequential frames of time - lapse microscopy of HeLa cells grown to confluence separated by a stopper . Stopper was removed at t0 and cellular migration was imaged for the indicated times . ( J ) Quantification of the % of wound area occupied by migrating cells over time stopper removal ( upper panel ) and respective rate of closure ( lower panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Wound closure assay. (I) Sequential frames of time-lapse microscopy of HeLa cells grown to confluence separated by a stopper. Stopper was removed at t0 and cellular migration was imaged for the indicated times. (J) Quantification of the % of wound area occupied by migrating cells over time stopper removal (upper panel) and respective rate of closure (lower panel).


( A ) Epifluorescence microscopy images of supernatants from LLO - treated ( 0 . 5 nM 15 min ) shCtlr , shGp96 or shNMHCIIA HeLa cells collected into poly - lysine - coated cover slips , fixed and stained with FITC - WGA ( green ) and DAPI ( blue ) . Insets show PM blebs of variable sizes and DAPI staining confirms the absence of cell nuclei . Scale bar - 10 μm . ( B ) Quantification of released PM blebs ( total FITCWGA / field ) . Values are the means ± SEM ( n ≥ 3 ) . * * * p < 0 . 001 .

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

(A) Epifluorescence microscopy images of supernatants from LLO-treated (0.5 nM 15 min) shCtlr, shGp96 or shNMHCIIA HeLa cells collected into poly-lysine-coated cover slips, fixed and stained with FITC-WGA (green) and DAPI (blue). Insets show PM blebs of variable sizes and DAPI staining confirms the absence of cell nuclei. Scale bar - 10 μm. (B) Quantification of released PM blebs (total FITCWGA/field). Values are the means ± SEM (n≥3). ***p<0.001.


( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( C ) 0 . 5 nM LLO for the indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (C) 0.5 nM LLO for the indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.


( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( D ) 1 . 5 or 3 nM LLO for 15 min ; or ( E ) 0 . 1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (D) 1.5 or 3 nM LLO for 15 min; or (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.


( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( E ) 0 . 1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.