Tandem ubiquitin binding entity ( TUBE ) pulldown of endogenous ubiquitylated ∆ Np63 in A - 431 upon DOX depletion of USP28 . Relative ubiquitination of representative immunoblot was calculated using ACTIN for normalization .

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

Tandem ubiquitin binding entity (TUBE) pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon DOX depletion of USP28. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.


Cycloheximide ( CHX ) chase ( 100µg / ml ) of control or inducible sh - USP28 A - 431 ( EtOH or 1µg / ml dox ) for indicated time points . Representative immunoblot ( left , VINCULIN as loading control ) of USP28 ∆ Np63 of relative abundance ( right ) .

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

Cycloheximide (CHX) chase assay (100µg/ml) of control or inducible sh-USP28 A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot (left, VINCULIN as loading control) of USP28 and ∆Np63 and quantification of relative protein abundance (right).


Doxycycline induced murine USP28 overexpression ( EtOH or 1ug / ml dox for 96 hours ) in A - 431 followed by immunoblot ( VINCULIN as loading control ) qPCR of USP28 ∆ Np63 . For qPCR , human USP28 murine USP28 ( mUSP28 ) primers were used . Relative mRNA was calculated using ∆ ∆ Ct for human USP28 ∆ Ct for mUSP28 ( ACTIN as housekeeping ) .

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

Doxycycline induced murine USP28 overexpression (EtOH or 1ug/ml dox for 96 hours) in A-431 cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63. For qPCR, human USP28 and murine USP28 (mUSP28) primers were used. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping).


TUBE pulldown of endogenous ubiquitylated ∆ Np63 in A - 431 upon overexpression of mUSP28 for 96 hours ( EtOH or 1µg / ml dox ) . Relative ubiquitination of representative immunoblot was calculated using ACTIN for normalization .

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

TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon overexpression of mUSP28 for 96 hours (EtOH or 1µg/ml dox). Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.


CHX chase ( 100µg / ml ) of control or inducible mUSP28 overexpressing A - 431 ( EtOH or 1µg / ml dox ) for indicated time points . Representative immunoblot of USP28 ∆ Np63 as well of relative abundance ( 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 B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

CHX chase assay (100µg/ml) of control or inducible mUSP28 overexpressing A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot analysis of USP28 and ∆Np63 as well quantification of relative protein abundance (VINCULIN as loading control).


Immunoblot of control ( sh - NTC ) two independent shRNA targeting USP28 ( sh - USP28 # 1 # 2 ) for ∆ Np63 USP28 abundance in LUDLU - 1adh ( VINCULIN as loading control ) , followed by qPCR of USP28 ∆ Np63 expression relative to ACTIN .

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

Immunoblot of control (sh-NTC) and two independent shRNA targeting USP28 (sh-USP28#1 and #2) for ∆Np63 and USP28 protein abundance in LUDLU-1adh (VINCULIN as loading control), followed by qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN.


Doxycycline induced mUSP28 overexpression ( EtOH or 1µg / ml dox for 96 hours ) in LUDLU - 1adh followed by immunoblot ( VINCULIN as loading control ) qPCR of USP28 , mUSP28 ∆ Np63 . Relative mRNA was calculated using ∆ ∆ Ct for human USP28 ∆ Ct for mUSP28 ( ACTIN as housekeeping for ) .

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

Doxycycline induced mUSP28 overexpression (EtOH or 1µg/ml dox for 96 hours) in LUDLU-1adh cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28, mUSP28 and ∆Np63. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping for the analysis).


Correlation of gene expression changes upon constitutive transduction of A - 431 with either shRNA targeting USP28 ( sh - USP28 # 1 ) or ∆ Np63 ( sh - ∆ Np63 # 2 ) relative to non - targeting control ( sh - NTC ) . diagonal reflects a regression build on a linear model . R : Pearsons correlation coefficient , m : slope of linear regression

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

Correlation of gene expression changes upon constitutive transduction of A-431 cells with either shRNA targeting USP28 (sh-USP28#1) or ∆Np63 (sh-∆Np63#2) relative to non-targeting control (sh-NTC). The diagonal line reflects a regression build on a linear model. R: Pearsons correlation coefficient, m: slope of the linear regression model


Gene set enrichment ( GSEA ) of a gene set of significantly down - regulated genes in sh - ∆ Np63 # 2 transfected A - 431 ( " Down - regulated sh - ∆ NP63 " , Appendix Table S1 ) . gene set was analysed in sh - ∆ Np63 # 2 ( left ) sh - USP28 # 1 - depleted ( right ) A - 431 . ( N ) ES : ( normalised ) enrichment

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

Gene set enrichment analysis (GSEA) of a gene set of significantly down-regulated genes in sh-∆Np63#2 transfected A-431 cells ("Down-regulated sh-∆NP63", Appendix Table S1). The gene set was analysed in sh-∆Np63#2 (left) and sh-USP28#1-depleted (right) A-431 cells. (N)ES: (normalised) enrichment score


Gene set enrichment ( GSEA ) of a gene set of significantly down - regulated genes in sh - USP28 # 1 transfected A - 431 ( " Down - regulated sh - USP28 " , Appendix Table S1 ) . gene set was analysed in shUSP28 # 1 ( left ) sh - ∆ Np63 # - depleted ( right ) A - 431 . ( N ) ES : ( normalised ) enrichment

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

Gene set enrichment analysis (GSEA) of a gene set of significantly down-regulated genes in sh-USP28#1 transfected A-431 cells ("Down-regulated sh-USP28", Appendix Table S1). The gene set was analysed in shUSP28#1 (left) and sh-∆Np63#-depleted (right) A-431 cells. (N)ES: (normalised) enrichment score


Immunoblot of endogenous ΔNp63 USP28 in A - 431 stably transduced with constitutive shRNA - non - targeting control ( NTC ) or against USP28 transiently transfected with exogenous ΔNp63 . ACTIN served as loading control . Representative western blot from three independent experiments .

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

Immunoblot of endogenous ΔNp63 and USP28 in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC) or against USP28 and transiently transfected with exogenous ΔNp63. ACTIN served as loading control. Representative western blot from three independent experiments.


growth of A - 431 stably transduced with constitutive shRNA - non - targeting control ( NTC ) or against USP28 transiently transfected with exogenous ΔNp63 . Total number was measured assessed at indicated time points . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) . p - values were calculated using two - t - statistical . * p - value < 0 . 05 , * * p - value < 0 .

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 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

Cell growth of A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC) or against USP28 and transiently transfected with exogenous ΔNp63. Total cell number was measured and assessed at indicated time points. Quantitative graph is represented as mean ± SD of three experiments (n=3). p-values were calculated using two-tailed t-test statistical analysis. *p-value < 0.05, **p-value < 0.01


Relative expression of SCC marker genes KRT5 , 14 19 in A - 431 stably transduced with constitutive shRNA - non - targeting control ( NTC ) , two independent shRNA - ΔNp63 , two independent shRNA - USP28 or ΔNp63 in shRNA - USP28 # 1 , normalised to ACTIN . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) .

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

Relative expression of SCC marker genes KRT5, 14 and 19 in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC), two independent shRNA-ΔNp63, two independent shRNA-USP28 or ΔNp63 in shRNA-USP28#1, normalised to ACTIN. Quantitative graph is represented as mean ± SD of three experiments (n=3).


Relative expression of epithelial marker genes KRT10 GPCR5A in A - 431 stably transduced with constitutive shRNA - non - targeting control ( NTC ) , two independent shRNA - ΔNp63 shRNA - USP28 # 1 , normalised to ACTIN . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) .

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

Relative expression of epithelial marker genes KRT10 and GPCR5A in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC), two independent shRNA-ΔNp63 and shRNA-USP28#1, normalised to ACTIN. Quantitative graph is represented as mean ± SD of three experiments (n=3).


Genomic signature of human lung SCC samples comprising USP28 , ∆ Np63 , KRT5 , KRT14 , KRT19 , DSG3 , MKI67 , PCNA GPRC5A . Samples were sorted dependent on relative USP28 expression ( high to low ) . n = 553 . Xena UCSC software .

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

Genomic signature of primary human lung SCC samples comprising USP28, ∆Np63, KRT5, KRT14, KRT19, DSG3, MKI67, PCNA and GPRC5A. Samples were sorted dependent on relative USP28 expression (high to low). n=553. Xena UCSC software.


GSEA of consensus squamous cancer marker genes ( se Appendix Table S2 ) in A - 431 stably transduced with sh - ∆ Np63 # 2 or sh - NTC . ( N ) ES : ( normalised ) enrichment score GSEA of consensus squamous cancer marker genes in A - 431 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 TP63 in lung SCC non - transformed lung tissue ( Normal ) . R : Spearmans correlation coefficient . n = 836 . Gepia software . Correlation of mRNA expression of consensus squamous cancer marker genes USP28 in lung SCC non - transformed lung tissue ( Normal ) . R : Spearmans correlation coefficient . N = 836 . Gepia software .

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

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 eosin ( H & E ) of tumour bearing 12 post intratracheal infection . Boxes indicate highlighted tumour areas in ( C ) ( a , b , a ' b ' ) . Scale bar = 2000µm ; nKPL = 6 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

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

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O 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.


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

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

O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 ) KPLU ( n = 5 ) upon AAV intratracheal infection . p - value was calculated using a log - rank .

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

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 murine KPSCC upon targeting with two independent shRNA sequences . ACTIN served as loading control .

O O O B-MEASURED_VAR 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 eosin ( H & E ) of tumour bearing 8 post intratracheal transplantation of 2 × 105 / . 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 B-CONTROLLED_VAR 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


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

O O O O O O 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

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

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


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

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

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

O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 TP63 in ADC SCC tumours for Lung ( nADC = 513 nSCC = 498 ) , Cervix ( nADC = 47 nSCC = 254 ) Oesophagus ( nADC = 89 nSCC = 96 ) . Blue dots : ADC ; Red dots : SCC ; R = Spearmans correlation coefficient ; m : Slope . Xena UCSC .

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

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

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

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).


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

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 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

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 upon treatment with warheads ( WH ) 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR 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 KRT14 in A - 431 treated for 24 hours with either DMSO or indicated concentrations of AZ1 . VINCULIN served as loading control . TP63 USP28 half maximal inhibitory abundance ( IC50 ) was calculated . Graphs are represented as mean ± SD ( n = 3 ) .

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

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 upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours . VINCULIN served as loading control for relative Ubiquitination .

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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 KRT14 in A - 431 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 .

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

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

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

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

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

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

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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 STAT expression 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 24 h . Whole extracts were collected tested in western blot for of phosphorylation of STAT1 ( Y701 ) STAT2 ( Y689 ) total level of STAT1 STAT2 . blots are representative of more than 3 independent experiments .

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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 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 48 h . Gene expression was measured by Q - PCR normalized to Gapdh to expression in untreated wild type . bars represent mean values with standard deviations ( SD ) of three independent experiments . C Effect of STAT1Y701Fheterozygosity on 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 48 h . Gene expression was measured by Q - PCR normalized to Gapdh to expression in untreated wild type . bars represent mean values with standard deviations ( SD ) of three independent experiments .

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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 organs . Spleens , livers or bone marrow derived macrophages ( BMDMs ) were isolated from wild type ( WT ) , Stat1Y701F ( YF ) Stat1 - / - ( S1 ) mice . Whole extracts were collected tested for of total Stat1 in western blot . blots are representative of more than 3 independent experiments .

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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 ) Stat1 - / - ( S1 ) mice stimulated for 30 min with 250 IU / mL of IFNβ or 5 ng / mL of IFNγ . Whole extracts were collected tested for of STAT1 phosphorylation on Y701 in western blot . blots are representative of more than 3 independent experiments .

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

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

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

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

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

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

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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 STAT2 with 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 . were crosslinked , sonicated immunoprecipitated with either STAT1 - specific antibody re - immunoprecipitated with STAT2 - specific antibody or vice versa . amount of precipitated was measured by Q - PCR . Bars represent mean values of three independent experiments ; error bars represent standard deviation ( SD ) .

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

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 indicated . 24 h after transfection , 250 IU / ml of IFNβ was added to transfected ISG expression was determined by Q - PCR after 48h of cytokine treatment . Gene expression was measured by Q - PCR normalized to Gapdh . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of mean ( ) asterisks denote level of statistical significance ( * , p ≤ 0 . 05 ) ; p - values were calculated using paired t - .

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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 of STAT2 translocation by immunofluorescence . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) IRF9 - / - ( IRF9 ) mice were seeded on cover slips stimulated with 250 IU / mL of IFNβ for 30 min or 24 h . were fixed 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 . scale bars represent 10 µm . B Quantitative evaluation of STAT2nuclear translocation . of STAT2 - dependent immunofluorescence over ( DAPI ) was quantified using ImageJ software in 20 from two independent experiments . Bars represent a mean with standard deviation ( SD ) asterisks denote level of statistical significance ( * * * , p ≤ 0 . 001 ) ; p - value was calculated using unpaired t - .

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

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 24 - well plates infected with L . pneumophila ( JR32 Fla - , MOI 0 . 25 ) . numbers of colony forming units ( CFUs ) were determined 24 h , 48 h 72 h after infection on charcoal yeast extract plates ( CYE ) . 0 time point was collected 1 . 5 h after 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 then infected with L . pneumophila ( JR32 Fla - , MOI 0 . 25 ) . numbers of colony forming units ( CFUs ) were determined 24 h , 48 h 72 h after infection on charcoal yeast extract plates ( CYE ) . 0 time point was collected 1 . 5 h after 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 of STAT1 tyrosine phosphorylation . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) Stat1 - / - ( S1 ) mice were infected with L . monocytogenes ( LO28 , MOI 10 ) for 5 or 6 h . Whole extracts were collected tested in western blot for of total STAT1 phosphorylation of STAT1 on tyrosine ( Y701 ) . 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 expression of IFNβ gene . BMDMs of wild type ( WT ) , Stat1Y701F , Stat1 - / - , Stat2 - / - IRF9 - / - mice were infected with L . monocytogenes ( LO28 , MOI 10 ) for 4 , 8 , 12 , 24 or 48 h . of Ifnβ gene expression were determined by Q - PCR . Bars represent mean values of three independent experiments . Error bars represent standard error of mean ( ) .

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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 growth of Listeria monocytogenes in macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F 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 . graph represents biological triplicates data are represented as mean values . Error bars represent standard deviation ( SD ) asterisks denote statistically significant differences ( ns , p > 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ) ; p - values were calculated using unpaired t - .

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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 Stat1 - / - mice per group were infected by intraperitoneal injection of 1x 102 viable L . monocytogenes . Survival was monitored over 10 days . 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 ) 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 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 median 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 .

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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 of infection inflammatory infiltrates . Wild type ( WT ) , Stat1Y701F 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 . scale bars on 20x magnification represent 100 µm 50 µm on 63x magnification . B Quantitative evaluation of inflammatory infiltrates . Infiltrates representing entire surface of sections from five per genotype were counted categorized according to their .

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

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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 in 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 in upstream downstream halves of endothelial in thoracic aorta of wild - type mouse . Each dot is one endothelial . Mean ± , n = 15 .

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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 laminar flow of 15 dyn / cm2 of shear force for 5 , 10 , 30 min . ( D ) of VE - PTP signals of shown in C , which co - localize with EEA1 . Mean ± , 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 , for VE - PTP ( red ) nuclei ( blue , Hoechst 33342 ) . ( F ) VE - PTP on surface measured by - ELISA of HUVEC after exposure as in C D . Red black lines show measurements with anti - VE - PTP antibody isotype control antibody . Mean ± , 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 ) at borders VE - PTP ( red ) downstream polarization in outer curvature in C in contrast to weak , dispersed for Tie2 - pY992 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 of Tie2 - pY992 in outer inner curvatures . ( F ) Tie2 - pY992 mean at junctions ( normalized to outer curvature = 1 . 0 ) . Mean ± , 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 intensities of extravasated antibody showing significantly stronger due to greater leakage in inner curvature . ( F ) Mean value for each mouse in two groups shown in E . Mean ± , 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 VE - PTP polarization in endothelial in thoracic aorta . Left right panels are same image with colors switched to highlight leakage ( red ) polarized VE - PTP ( green , arrowheads ) on left , polarized VE - PTP ( red , arrowheads ) , VE - cadherin ( green ) , leakage ( blue ) on right .

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(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 at intercellular junctions in aortic arch endothelial showing lower values in inner curvature than in 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 ± , n = 6 mice / group .

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(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 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 lysates ( bottom ) immunoblotted for pan - phosphotyrosine ( antibody 4G10 ) , Tie2 , or VE - PTP . of for phosphorylated Tie2 total Tie2 ( Tie2 - pY to Tie2 , n = 4 experiments ) . Right . Relative Tie2 - pY in HUVEC after control siRNA or VE - PTP siRNA . Normalized to static control = 1 .

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

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(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 for VE - cadherin ( green ) . ( F G ) of Streptavidin leakage per image . Mean ± , n = 19 pooled in ten independent experiments ( F ) , n = 6 pooled in three independent experiments ( G ) .

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

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

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(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 at intercellular junctions in aortic arch of mice shown in B . Mean ± , n = 8 mice / group .

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

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(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 stained with DAPI ( blue ) , LDHA ( green ) , LDHB ( red ) . Magnification boxes show different areas as depicted in main image . of LDHA LDHB was performed on spheroid areas as indicated in graphs . Scale bar : 100 µm . Data are represented as mean ± s . d . ( 5 - 8 fields per 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 B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of LDHA LDHB expression in P3 transduced with CRISPR - Cas9 lentiviral vectors with scramble ( sgControl ) or against LDHA ( sgLDHA ) , LDHB ( sgLDHB ) or both ( sgLDHA / B ) . Knockout ( KO ) were exposed to 21 % or 1 % O2 for 48 h .

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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 analyzed using Kruskal - Wallis followed by Dunn ' s multiple comparisons . Spheroid growth : sgCont vs sgLDHA , P = 0 . 0003 ; sgCont vs sgLDHB , P = 0 . 78 ; sgCont vs sgLDHA / B , P < 0 . 0001 . Dead : 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 .

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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 analyzed using Two - way ANOVA following by Tukey ' s multiple comparisons : 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 . 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 B-CONTROLLED_VAR 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 stained . For second cohort , each mouse was sacrificed when it reached a limit point allowing draw of survival curves . Left : Immunofluorescence of Nestin ( top middle ) CD31 ( bottom ) of control LDHA / B KO P3 section . Scale bars : 2 mm ( top ) , 250 µm ( middle ) 100 µm ( bottom ) . Right : Graphs represent invasion of control LDHA / B KO P3 tumors in mm2 ( n = 5 mice per group , average of 5 - 6 sections per ) . Data are represented as mean . For F , data are analyzed using One - way ANOVA following by Dunnett ' s multiple comparisons . : 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 . : sgCont vs sgLDHA / B , P = 0 . 005 . Invasion : sgCont vs sgLDHA / B , P = 0 . 01 .

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

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

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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 , treated with phenformin or irradiated with 10 Gy ( n = 6 - 8 mice per group ) . Data are analyzed using Log - rank ( Mantel - Cox ) : 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 .

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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 . growth at 72 h are analyzed using unpaired t : 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 analyzed using unpaired t : P < 0 . 0001 . Right : Representative of invasive spheroids for each condition . Scale bar : 100 µm .

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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 . Mice were treated either with vehicle ( blue ) or stiripentol ( red ) ( n = 15 - 16 mice per group ) . Data are analyzed using Log - rank ( Mantel - Cox ) : P = 0 .

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

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

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

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

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

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

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(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 of LLO - treated HeLa 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 .

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

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(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 of HeLa left untreated ( Control ) or treated with LLO ( 0 . 5 nM 15 min ) . were stained with FITC - WGA ( Plasma , PM - red ) immunolabelled for NMHCIIA ( green ) . Insets show PM blebs arrows indicate recruitment of NMHCIIA bundles to PM blebs associated ( 1 ) or detached ( 2 ) from .

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

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

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

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 of HeLa treated as indicated immunolabelled for ER - KDEL ( blue ) , NMHCIIA ( green ) Gp96 ( red ) .

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


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

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(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 Gp96 from Gp96 IP of HeLa 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 B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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 O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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).