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 - .

O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ( ) .

O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

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-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 - .

O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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 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-GENEPROD O O B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY 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-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

O O O B-GENEPROD O O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM O O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD O O O B-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-TISSUE O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O 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 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 ) . (

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O

(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 .

O O O B-EXP_ASSAY O O O O O O O O B-TISSUE O O B-TISSUE I-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 .

O O O O O B-GENEPROD O O O O O O B-TISSUE I-TISSUE B-CELL_TYPE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM 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 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 .

O O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 ) .

O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 ) .

O O O B-CELL_TYPE O O B-EXP_ASSAY B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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 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 .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY B-DISEASE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM 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 ) 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 .

O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM 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 at intercellular junctions in aortic arch of mice shown in B . Mean ± , n = 8 mice / group .

O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE I-TISSUE O B-ORGANISM O O O O O O O O O O B-ORGANISM 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 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 .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY B-DISEASE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM 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 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 B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O 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-GENEPROD O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 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 .

O O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD 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 .

O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O 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-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY 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 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE 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 ) 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 .

O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY B-SUBCELLULAR O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR 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 , 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O 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 . 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 .

O O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-ORGANISM O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM 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 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 ) .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD 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 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD 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 ) .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O B-CELL_LINE O O O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR 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 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

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR 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 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 )

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD 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 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 )

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD 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 of HeLa treated with aerolysin immunolabelled for NMHCIIA ( green ) Gp96 ( red ) . Insets show NMHCIIA bundles arrows indicate association between Gp96 NMHCIIA .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD 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 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD 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 of shCtrl , shGp96 or shNMHCIIA HeLa treated with LLO ( 0 . 5 nM , 15 min ) immunolabelled for KDEL - ( blue ) , NMHCIIA ( green ) Gp96 ( red ) . Insets show compact NMHCIIA bundles in shCtrl dispersed NMHCIIA bundles in shGp96 . Scale bar - 10 μm . ( B - D ) of % of 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 mean ± ( n ≥ 3 ) , p - values were calculated using ( B - C ) one - way - ANOVA with Dunnett post hoc analyses ( D ) two - un - paired Student ' s t - , * p < 0 . 5 , * * * p < 0 . 001 . For shNMHCIIA , bundles were detected following actin ( Fig EV3C ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 or ( F ) retracting blebs per evaluated by time - lapse microscopy of LLO - treated shCtrl or shGp96 . shCtrl n = 32 shGp96 n = 40 , p - values were calculated using two - un - paired Student ' s t - * p < 0 . 5 . ( G ) Sequential frames of time - lapse microscopy of LLO - treated HeLa expressing GFP - NMHCIIA ( shCtrl , shGp96 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 B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD 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 actin from shCtrl or shGp96 left untreated ( U ) or treated with 0 . 1 nM LLO for indicated time points . of pMRLC corresponds to mean ± ( 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 Fig EV3 .

O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of LLO - treated HeLa immunolabelled for indicated . Insets show colocalization between NMHCIIA WRAMP components at cortical bundles . ( Aii ) of Pearson ' s correlation coefficient ( PCC ) between NMHCIIA indicated within entire LLO - treated , cortical bundles or equivalent control ROI ( Ctrl ROI ) . For Calpain 2 cortical bundles were defined by actin . 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD I-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Filamin A from WCL Gp96 IP of HeLa left untreated ( U ) or treated with LLO ( 0 . 5 nM , 15 min ) ( LLO ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD 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 Gp96 from WCL Gp96 IP of HeLa left untreated ( U ) or treated with 25 μg / ml of Wnt5a for indicated time .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD 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 of HeLa untreated ( Mock ) or treated with 25 μg / ml of Wnt5a for 30 min . were immunolabelled for ER - KDEL ( blue ) , Gp96 ( red ) NMHCIIA ( green ) . Arrows indicate polarised localization of NMHCIIA , ER - KDEL Gp96 in Wnt5a - treated .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O B-GENEPROD 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 of shCtlr , shGp96 or shNMHCIIA HeLa stained for actin ( red ) DAPI ( blue ) 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 arrows indicate stress or distinct focal adhesion points . of % of with ( G ) stress or ( H ) focal adhesion points labelled by FAK .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD 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 . ( I ) Sequential frames of time - lapse microscopy of HeLa grown to confluence separated by a stopper . Stopper was removed at t0 migration was imaged for indicated times . ( J ) of % of wound occupied by migrating over time stopper removal ( upper panel ) respective rate of closure ( lower panel ) .

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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O 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 of % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa . were treated with ( C ) 0 . 5 nM LLO for indicated times . of PI incorporation by untreated were subtracted from LLO - treated samples . Values are mean ± ( n ≥ 4 ) 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa . 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 recovery for indicated times . of PI incorporation by untreated were subtracted from LLO - treated samples . Values are mean ± ( n ≥ 4 ) 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa . were treated with ( E ) 0 . 1 nM LLO for 10 min followed by LLO washed out recovery for indicated times . of PI incorporation by untreated were subtracted from LLO - treated samples . Values are mean ± ( n ≥ 4 ) 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


( A - B ) Epifluorecence microscopy of HeLa ( A ) left untreated or treated with LLO ( 0 . 5 nM , 15 min ) . were incubated with exofacial - PS probe ( Alexa - 568 annexin A5 ) ( red ) 30 min prior fixation , immunolabelled for NMHCIIA ( green ) stained with DAPI ( blue ) . Insets show PM damage marked by exofacial - PS sites . Arrows indicate exofacial - PS associated with intracellular in shCtrl with NMHCIIA bundles ; arrowheads indicate NMHCIIA bundles associated with exofacial - PS sites without detectable .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O

(A-B) Epifluorecence microscopy images of HeLa cells (A) left untreated or treated with LLO (0.5 nM, 15 min). Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.


( A - B ) Epifluorecence microscopy of HeLa ( B ) infected with GFP - expressing wt Lm for 6 h . were incubated with exofacial - PS probe ( Alexa - 568 annexin A5 ) ( red ) 30 min prior fixation , immunolabelled for NMHCIIA ( green ) stained with DAPI ( blue ) . Insets show PM damage marked by exofacial - PS sites . Arrows indicate exofacial - PS associated with intracellular in shCtrl with NMHCIIA bundles ; arrowheads indicate NMHCIIA bundles associated with exofacial - PS sites without detectable .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O

(A-B) Epifluorecence microscopy images of HeLa cells (B) infected with GFP-expressing wt Lm for 6 h. Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.


( C ) of % of infected with exofacial - PS sites in HeLa infected with wt Lm for 6 h . Data are mean ± ( n = 3 ) p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 .

O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O B-ORGANISM O O O O O O O O O O O O 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) Quantification of the % of infected cells with exofacial-PS sites in HeLa cells infected with wt Lm for 6 h. Data are the mean ±SEM (n=3) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5.


( D - E ) Flow cytometry of % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa RAW264 . 7 , infected with GFP - expressing wt or Δhly Lm for 6 h . of PI incorporation by uninfected were subtracted from infected samples . Values are mean ± ( n ≥ 4 ) p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . PM permeability was assessed in harbouring equivalent numbers of intracellular ( Fig Appendix Fig S3 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O

(D-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa and RAW264.7 cells, infected with GFP-expressing wt or Δhly Lm for 6 h. The levels of PI incorporation by uninfected cells were subtracted from infected 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. PM permeability was assessed in cells harbouring equivalent numbers of intracellular bacteria (Fig Appendix Fig S3).


( A ) Immunoblots of NMHCIIA Gp96 from whole lysates ( WCL ) IP fractions ( IP Gp96 ) of extracts of zebrafish larvae 3 dpf uninfected ( U ) or infected with wt or ∆ hly Lm ( low dose 200 - 1500 CFU ) for 24h .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O

(A) Immunoblots of NMHCIIA and Gp96 levels from whole cell lysates (WCL) and IP fractions (IP Gp96) of extracts of zebrafish larvae 3 dpf uninfected (U) or infected with wt or ∆hly Lm (low dose 200-1500 CFU) for 24h.


( B ) Immunoblots of Gp96 tubulin from zebrafish 3 dpf larvae injected with control ( Ctrl - mo ) or Gp96 morpholino oligonucleotides ( Gp96 mo ) .

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(B) Immunoblots of Gp96 and tubulin levels from zebrafish 3 dpf larvae injected with control (Ctrl-mo) or Gp96 morpholino oligonucleotides (Gp96 mo).


( C ) Confocal microscopy of zebrafish larvae ( Ctrl mo or Gp96 mo ) infected ( low dose ) in tail muscle , with GFP - expressing wt or Δhly Lm for 24 h , immunolabelled for KDEL - ( green ) , stained with phalloidin ( actin , red ) DAPI ( ) . show actin rich structures at cortex ( arrows ) ER - KDEL ( arrowheads ) in larvae infected with wt .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM O O O O B-GENEPROD O O O O O O O O B-TISSUE I-TISSUE O O B-GENEPROD O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-GENEPROD O O O O B-ORGANISM O O O O

(C) Confocal microscopy images of zebrafish larvae (Ctrl mo or Gp96 mo) infected (low dose) in the tail muscle, with the GFP-expressing wt or Δhly Lm for 24 h, immunolabelled for KDEL-proteins (green), and stained with phalloidin (actin, red) and DAPI (white). Images show actin rich structures at the cell cortex (arrows) and ER-KDEL puncta (arrowheads) in larvae infected with wt strain.


( D ) CFU per zebrafish larvae ( Ctrl or Gp96 mo ) infected with wt ( low dose ) or Δhly ( high dose > 10 , 000 CFU ) Lm analysed at 0 , 24 , 48 hpi . Results are mean ± ( n ≥ 3 ) ( horizontal bars ) each circle represents 1 larvae . p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 .

O O O O O B-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O 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-ORGANISM O 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) CFU counts per zebrafish larvae (Ctrl or Gp96 mo) infected with wt (low dose) or Δhly (high dose > 10,000 CFU) Lm and analysed at 0, 24, 48 hpi. Results are mean ± SEM (n≥3) (horizontal bars) and each circle represents 1 larvae. p-values were calculated using one-way-ANOVA with Tukey post hoc analyses **p<0.01.


( E ) Survival curves of zebrafish larvae ( Ctrl - mo or Gp96 - mo ) infected with a wt ( low dose ) or Δhly ( high dose > 10 , 000 CFU ) Lm . Results are mean ± ( n ≥ 3 ) . wt infection of Ctrl Gp96 mo , n = 28 larvae ; Δhly infection of Gp96 mo n = 13 . p - values were calculated using Log Rank , * * p < 0 . 01 .

O O O B-EXP_ASSAY O O B-ORGANISM I-ORGANISM O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(E) Survival curves of zebrafish larvae (Ctrl-mo or Gp96-mo) infected with a wt (low dose) or Δhly (high dose > 10,000 CFU) Lm. Results are the mean ± SEM (n≥3). wt infection of Ctrl and Gp96 mo, n=28 larvae; Δhly infection of Gp96 mo n=13. p-values were calculated using Log Rank test, **p<0.01.


( F ) CFU per zebrafish larvae infected with a high dose of ∆ hly Lm at different times post - infection . Results are mean ± ( n ≥ 3 ) ( horizontal bars ) ; each point represents 1 larvae . See also Fig EV5 .

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(F) CFU counts per zebrafish larvae infected with a high dose of ∆hly Lm at different times post-infection. Results are mean ± SEM (n≥3) (horizontal bars); each point represents 1 larvae. See also Fig EV5.


( F ) Examples of expression of Treg signature genes Foxp3 , Il10ra , Nrp1 , Ikzf4 Ccr6 ; examples of expression of genes upregulated in Tet2 / 3 DKO Treg Eomes , Ccl4 , Chek1 Fn1 . Y - axis shows FPKM ( Fragment Per Kilobase per Million reads ) , X - axis shows different types . Error bars show mean ± S . D . from two ( CD4 + naïve T ) or four ( WT Tet2 / 3 DKO Treg ) biological replicates .

O O O O O B-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-CELL_TYPE B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O 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-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O

(F) Examples of expression levels of Treg signature genes Foxp3, Il10ra, Nrp1, Ikzf4 and Ccr6; and examples of expression levels of genes upregulated in Tet2/3 DKO Treg cells Eomes, Ccl4, Chek1 and Fn1. The Y-axis shows FPKM (Fragment Per Kilobase per Million reads), the X-axis shows the different cell types. Error bars show mean ± S.D. from two (CD4+ naïve T cells) or four (WT and Tet2/3 DKO Treg) biological replicates.


( D ) Genome browser view of CMS - IP data showing CMS ( 5hmC ) distribution at Il2ra Foxp3 locus for CD4 + naïve T WT Treg . Hydroxymethylated regions ( hmRs ) DhmRs are shown on top ; conservation ( cons ) is shown at bottom .

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(D) Genome browser view of CMS-IP data showing CMS (5hmC) distribution at Il2ra and Foxp3 locus for CD4+ naïve T cells and WT Treg cells. Hydroxymethylated regions (hmRs) and DhmRs are shown on the top; conservation (cons) is shown at the bottom.


( E ) Genome browser view of methylation patterns of Hopx Il10 loci for CD4 + naïve T , WT Treg Tet2 / 3 DKO Treg . Differentially hydroxymethylated regions ( DhmRs ) differentially methylated CpGs ( DM - CpGs ) are shown on top of tracks . Conservation track ( Cons ) denotation of CpGs are shown at bottom . WGBS data are from two biological replicate samples .

O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O 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-EXP_ASSAY O O O O O O O O

(E) Genome browser view of DNA methylation patterns of Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. Differentially hydroxymethylated regions (DhmRs) and differentially methylated CpGs (DM-CpGs) are shown on top of the tracks. Conservation track (Cons) and denotation of all CpGs are shown at the bottom. WGBS data are from two biological replicate samples.


( E ) Genome browser view of accessibilities at Hopx Il10 loci for CD4 + naïve T , WT Treg Tet2 / 3 DKO Treg . DhmRs , DM - CpGs DARs ( shaded ) are shown on top of tracks . Conservation track ( Cons ) is shown at bottom .

O O O O O O O O O B-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE B-GENEPROD O O O B-CELL_TYPE O O O 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) Genome browser view of chromatin accessibilities at Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. DhmRs, DM-CpGs and DARs (shaded) are shown on top of the tracks. Conservation track (Cons) is shown at the bottom.


( H - J ) Box - - whisker plots of methylation for DM - CpGs less methylated in TGFβ + RA + VitC iTregs vs TGFβ iTregs ( H ) , DM - CpGs less methylated in WT Treg vs CD4 + naïve T ( I ) DM - CpGs less methylated in WT Treg vs Tet2 / 3 DKO Treg ( J ) . DM - CpGs denotes CpGs in respective types used for comparison . Boxplots show median quartiles with whiskers extending to most extreme data point within 1 . 5 times interquartile range . P - values are shown above relevant comparisons are calculated using an of variances with Tukey ' s post - hoc . Data are from two biological replicates .

O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-CELL_TYPE O B-GENEPROD B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Box-and-whisker plots of DNA methylation levels for DM-CpGs less methylated in TGFβ + RA + VitC iTregs vs TGFβ iTregs (H), DM-CpGs less methylated in WT Treg cells vs CD4+ naïve T cells (I) and DM-CpGs less methylated in WT Treg cells vs Tet2/3 DKO Treg cells (J). DM-CpGs denotes the CpGs in the respective cell types used for the comparison. Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. P-values are shown above relevant comparisons and are calculated using an analysis of variances with Tukey's post-hoc test. Data are from two biological replicates.


( D ) Graphs depicting of viable for TGFβ iTregs ( left ) TGFβ + RA + VitC iTregs ( right ) differentiated with 0 U / ml , 1 U / ml , 5 U / ml 10 U / ml IL - 2 . Error bars show mean ± S . D . from three biological replicates .

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(D) Graphs depicting the percentage of viable cells for TGFβ iTregs (left) and TGFβ+RA+VitC iTregs (right) differentiated with 0 U/ml, 1 U/ml, 5 U/ml and 10 U/ml IL-2. Error bars show mean ± S.D. from three biological replicates.


( C ) ChIP - qPCR showing STAT5 occupancy at Foxp3 CNS2 IL2Rα IN1b regions in TGFβ TGFβ + RA + VitC iTregs without or with 3 U / ml IL - 2 restimulation . GMPR is a negative control region . Data are from two biological replicates .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O

(C) ChIP-qPCR showing STAT5 occupancy at Foxp3 CNS2 and IL2Rα IN1b regions in TGFβ and TGFβ + RA + VitC iTregs without or with 3 U/ml IL-2 restimulation. GMPR is a negative control region. Data are from two biological replicates.


( D , E ) Imaging of luciferase activity in liver of HIF1aAlbKO ( D ) HIF2aAlbKO ( E ) mice after a hydrodynamic tail vein injection with a HRE - luc reporter plasmid or control ( PBS ) according to followed by 6h normoxia or hypoxia . ( F , G ) Bioluminescent photon normalized to PBS control group in liver of ( F ) HIF1aAlbKO ( G ) HIF2aALbKO mice their wild - type 6h after normoxia of hypoxia . Data information : bars represent mean ± . Each data point represent mice . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons to correct for multiple testing during pairwise multiple comparisons , except if otherwise stated . * * * P < 0 . 001 ; * * P ≤ 0 . 01 ; * P ≤ 0 . 05 . N = normoxia , H = hypoxia . Signal around perimeter of Petri dishes represent aspecific luciferase signal .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-TISSUE O O O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Imaging of luciferase activity in the liver of HIF1aAlbKO (D) and HIF2aAlbKO (E) mice after a hydrodynamic tail vein injection with a HRE-luc reporter plasmid or control (PBS) according to body weight followed by 6h normoxia or hypoxia. (F, G) Bioluminescent photon counts normalized to the PBS control group in liver of (F) HIF1aAlbKO and (G) HIF2aALbKO mice and their wild-type littermates 6h after normoxia of hypoxia. Data information: All bars represent mean ± SEM. Each individual data point represent individual mice. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. *** P<0.001; ** P≤ 0.01; * P≤ 0.05. N = normoxia, H = hypoxia. Signal around the perimeter of the Petri dishes represent aspecific luciferase signal.


( E ) Scatter plot showing log fold change ( LFC ) of DEX - upregulated genes ( LFC > 1 P ≤ 0 . 05 ) DEX - downregulated genes ( LFC < - 1 P ≤ 0 . 05 ) in normoxia vs hypoxia after 24h . black represents diagonal , red represents real slope ± standard error of data as analysed by linear regression .

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(E) Scatter plot showing log fold change (LFC) of all DEX-upregulated genes (LFC > 1 and P ≤ 0.05) and DEX-downregulated genes (LFC < -1 and P ≤ 0.05) in normoxia vs hypoxia after 24h. The black line represents the diagonal, and the red line represents the real slope ± standard error of the data as analysed by linear regression.


( F , G ) Examples of GR - responsive genes on - seq data 6h ( F ) 24h ( G ) after normoxia or hypoxia DEX stimulation . Data information : bars represent mean ± . N = 3 per group . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons to correct for multiple testing during pairwise multiple comparisons , except if otherwise stated . * * * P < 0 . 001 , * * P < 0 . 01 .

O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Examples of GR-responsive genes based on the RNA-seq data 6h (F) and 24h (G) after normoxia or hypoxia and DEX stimulation. Data information: All bars represent mean ± SEM. N=3 per group. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01.


( H ChIP - seq on liver derived from mice which were subjected to hypoxia ( 6h 24h ) , followed by DEX ( 10 mg / kg ) injection 2h later sacrificed for liver isolation . ( H ) Box plots showing LFC of genes responsive to DEX in both normoxia or hypoxia ( 6h 24h ) . central band represents median . box ranges from first quartile ( Q1 ) to third quartile ( Q3 ) which represents interquartile range ( IQR = Q3 - Q1 ) covers central 50 % of data . whiskers illustrate minimum ( Q1 - 1 . 5 * IQR ) maximum ( Q3 + 1 . 5 * IQR ) of data . Outliers are shown as dots . N = 3 biological replicates per condition .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ChIP-seq on liver derived from mice which were subjected to hypoxia (6h and 24h), followed by DEX (10 mg/kg) injection and 2h later sacrificed for liver isolation. (H) Box plots showing the LFC of genes responsive to DEX in both normoxia or hypoxia (6h and 24h). The central band represents the median. The box ranges from the first quartile (Q1) to the third quartile (Q3) which represents the interquartile range (IQR = Q3 - Q1) and covers the central 50% of the data. The whiskers illustrate the minimum (Q1 - 1.5*IQR) and maximum (Q3 + 1.5*IQR) of the data. Outliers are shown as dots. N=3 biological replicates per condition.


C57BL / 6J mice were put in normoxia or hypoxia for 6h or 24h , injected i . p . with PBS or DEX ( 10 mg / kg ) livers were isolated for genome - wide transcriptomics via - seq . N = 3 per group for a single - seq . ( A , B ) Examples of DEX - responsive genes in hypoxia 6h ( A ) 24h ( B ) which were not induced by DEX in normoxia . Data information : bars represent mean ± . P - values were calculated using two - way ANOVA followed by post - hoc Šídák ' s multiple comparisons to correct for multiple testing during pairwise multiple comparisons , except if otherwise stated . * * * P < 0 . 001 , * * P < 0 . 01 , * P ≤ 0 . 05 .

O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Female C57BL/6J mice were put in normoxia or hypoxia for 6h or 24h, injected i.p. with PBS or DEX (10 mg/kg) and livers were isolated for genome-wide transcriptomics via RNA-seq. N=3 per group for a single RNA-seq. (A, B) Examples of DEX-responsive genes in hypoxia 6h (A) and 24h (B) which were not induced by DEX in normoxia. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01, *P≤0.05.