( A ) XPO1 and STK38 are required for YAP1 nuclear export at high confluence . A549 cells were transiently transfected with the indicated siRNA ( control and KPT conditions = siNT ) at low or high confluency . 48 hours later , cells were incubated overnight in the presence of XPO1 inhibitors KPT - 185 and KPT - 330 ( final concentration = 1 µM ) or with DMSO for all other conditions . The next day , cells were fixed and stained for endogenous YAP1 . Quantitative representation of YAP1 nuclear fluorescence intensity ( n > 300 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , Mann - Whitney test ) . XPO1 activity inhibition by both KPT - 185 and KPT - 330 as well as STK38 silencing failed to induce YAP1 nuclear exit at high

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

(A) XPO1 and STK38 are required for YAP1 nuclear export at high confluence. A549 cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT) at low or high confluency. 48 hours later, cells were incubated overnight in the presence of XPO1 inhibitors KPT-185 and KPT-330 (final concentration = 1 µM) or with DMSO for all other conditions. The next day, cells were fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n > 300 cells from 3 independent experiments, mean ± SEM; ***p<0.001, Mann-Whitney test). XPO1 activity inhibition by both KPT-185 and KPT-330 as well as STK38 silencing failed to induce YAP1 nuclear exit at high confluence


( B ) XPO1 _ S1055 phosphorylation is required and sufficient for YAP nuclear exit . Genome edited XPO1 HAP1 cells were cultured for two days at low versus high confluency . Cells were then fixed and stained for endogenous YAP1 . Quantitative representation of YAP1 nuclear fluorescence intensity ( n > 300 cells from 3 independent experiments , mean ± SEM ; * * * p < 0 . 001 , Mann - Whitney test ) . Here , phosphonegative XPO1 failed to induce YAP nuclear exit at high confluence whereas phosphomimetics variant ( S1055D & S1055E ) potentiated YAP1 nuclear

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(B) XPO1_S1055 phosphorylation is required and sufficient for YAP nuclear exit. Genome edited XPO1 HAP1 cells were cultured for two days at low versus high confluency. Cells were then fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n > 300 cells from 3 independent experiments, mean ± SEM; ***p<0.001, Mann-Whitney test). Here, phosphonegative XPO1 failed to induce YAP nuclear exit at high confluence whereas phosphomimetics variant (S1055D & S1055E) potentiated YAP1 nuclear exit


( C ) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 ( siSTK38 # 206 or with non - targeting siRNA ( siNT ) ) at low or high confluence . 72 hours later , cells were fixed and stained for endogenous YAP1 . Quantitative representation of YAP1 nuclear fluorescence intensity ( n = 150 cells from 2 independent experiments ) . Here , phosphomimetic XPO1 ( S1055D ) potentiated YAP1 nuclear exit at both low and high confluences even in the absence of STK38 as compared to cells carrying wt

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(C) Genome edited XPO1 mutant HAP1 cells were transiently transfected with siRNA targeting endogenous STK38 (siSTK38#206 or with non-targeting siRNA (siNT)) at low or high confluence. 72 hours later, cells were fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n = 150 cells from 2 independent experiments). Here, phosphomimetic XPO1 (S1055D) potentiated YAP1 nuclear exit at both low and high confluences even in the absence of STK38 as compared to cells carrying wt XPO1


( A ) Heatmap showing viability measurements for the 192 samples ( rows ) subject to each of 17 microenvironmental stimuli ( columns ) . The data are shown on a robust z - score scale , i . e . , the logarithm of the relative viability measurements were scaled by the median absolute deviation within each row . Limits were applied to scaling factor for optimal visualisation . Red indicates increased viability , blue indicates decreased viability . Samples are annotated for genetic features , sex and pre - treatment status .

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(A) Heatmap showing viability measurements for the 192 samples (rows) subject to each of 17 microenvironmental stimuli (columns). The data are shown on a robust z-score scale, i.e., the logarithm of the relative viability measurements were scaled by the median absolute deviation within each row. Limits were applied to scaling factor for optimal visualisation. Red indicates increased viability, blue indicates decreased viability. Samples are annotated for genetic features, sex and pre-treatment status.


Logarithm of the relative viability after treatment with Resiquimod ( A , n = 169 ) , IL4 ( B , n = 174 ) stratified by trisomy 12 , and in A , also by IGHV status . P - values from Student ' s t - test ( two - sided , non - paired ) . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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Logarithm of the relative viability after treatment with Resiquimod (A, n=169), IL4 (B, n=174) stratified by trisomy 12, and in A, also by IGHV status. P-values from Student's t-test (two-sided, non-paired). The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


Logarithm of the relative viability after treatment with TGFβ ( C , n = 174 ) stratified by trisomy 12 , P - values from Student ' s t - test ( two - sided , non - paired ) . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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Logarithm of the relative viability after treatment with TGFβ (C, n=174) stratified by trisomy 12, P-values from Student's t-test (two-sided, non-paired). The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


( A ) Graphical representation of the four drug - stimulus interaction categories . Categories are defined according to the nature of the interaction ( synergistic or antagonistic ) , and whether the viability is increased or decreased by the stimulus ( positive or negative ) . x - axis shows treatment type , y - axis shows viability with each treatment . Red and blue points and lines depict a representative treatment response pattern for given interaction type . Blue horizontal lines represent the expected viability for combinatorial treatment in the absence of an interaction between drug - stimulus ( i . e . additive effects ) , black horizontal lines represent the measured viability after combinatorial treatment . The difference between the black and blue lines indicates the impact of the interaction on expected viability .

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(A) Graphical representation of the four drug-stimulus interaction categories. Categories are defined according to the nature of the interaction (synergistic or antagonistic), and whether the viability is increased or decreased by the stimulus (positive or negative). x-axis shows treatment type, y-axis shows viability with each treatment. Red and blue points and lines depict a representative treatment response pattern for given interaction type. Blue horizontal lines represent the expected viability for combinatorial treatment in the absence of an interaction between drug-stimulus (i.e. additive effects), black horizontal lines represent the measured viability after combinatorial treatment. The difference between the black and blue lines indicates the impact of the interaction on expected viability.


( D - I ) Examples of drug - stimulus interactions , for each category . Plots show the natural logarithm of the relative viability of 192 CLL samples after treatment with the respective drug and stimulus . Each line represents one patient sample linked across treatments . Black horizontal lines in each single treatment indicate the expected viability based on linear modelling . Black and blue horizontal lines in the combinatorial treatment indicate the expected viability based on the additive effect of the drug and stimulus ( blue ) , and the expected viability accounting for the additive effect and the interaction ( black ) . ( D + E ) Ibrutinib , a clinically used BTK inhibitor , is blocked by IL4 and IFNγ . ( F ) The JAK inhibitor pyridone - 6 inhibits the pro - survival effect of sCD40L + IL4 stimulation . ( G ) The p38 inhibitor ralimetinib and IFNγ show a synergistic pro - survival effect not observed in either single treatment . ( H ) TLR agonists , including CpG ODN ( shown ) increase sensitivity to BTK inhibition by ibrutinib , despite increasing viability as single treatments . ( I ) Soluble anti - IgM sensitises CLL samples to HSP90 inhibition by luminespib .

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

(D - I) Examples of drug-stimulus interactions, for each category. Plots show the natural logarithm of the relative viability of 192 CLL samples after treatment with the respective drug and stimulus. Each line represents one patient sample linked across treatments. Black horizontal lines in each single treatment indicate the expected viability based on linear modelling. Black and blue horizontal lines in the combinatorial treatment indicate the expected viability based on the additive effect of the drug and stimulus (blue), and the expected viability accounting for the additive effect and the interaction (black). (D+E) Ibrutinib, a clinically used BTK inhibitor, is blocked by IL4 and IFNγ. (F) The JAK inhibitor pyridone-6 inhibits the pro-survival effect of sCD40L + IL4 stimulation. (G) The p38 inhibitor ralimetinib and IFNγ show a synergistic pro-survival effect not observed in either single treatment. (H) TLR agonists, including CpG ODN (shown) increase sensitivity to BTK inhibition by ibrutinib, despite increasing viability as single treatments. (I) Soluble anti-IgM sensitises CLL samples to HSP90 inhibition by luminespib.


Beeswarm - boxplots of the natural logarithm of the relative viability of 169 CLL samples , for ( C ) fludarabine + CpG ODN and combinatorial treatments , faceted by IGHV status and trisomy 12 status . P - values from paired Student ' s t - test . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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Beeswarm-boxplots of the natural logarithm of the relative viability of 169 CLL samples, for (C) fludarabine + CpG ODN and combinatorial treatments, faceted by IGHV status and trisomy 12 status. P-values from paired Student's t-test . The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


Beeswarm - boxplots of the natural logarithm of the relative viability of 169 CLL samples , for ( D ) ibrutinib + IL4 single and combinatorial treatments , faceted by IGHV status and trisomy 12 status . P - values from paired Student ' s t - test . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

Beeswarm-boxplots of the natural logarithm of the relative viability of 169 CLL samples, for (D) ibrutinib + IL4 single and combinatorial treatments, faceted by IGHV status and trisomy 12 status. P-values from paired Student's t-test . The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


( A Mean ( A ) pSTAT6 staining intensity in CLL - infiltrated ( n = 100 for pSTAT and tumour - free lymph node biopsies ( n = 98 for pSTAT6 after background subtraction ( y axis ) , p - values from Student ' s t - test . Each dot represents the mean of all cells in the tissue microarray cores per patient sample . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

(A Mean (A) pSTAT6 staining intensity in CLL-infiltrated (n=100 for pSTAT and tumour-free lymph node biopsies (n=98 for pSTAT6 after background subtraction (y axis), p-values from Student's t-test. Each dot represents the mean of all cells in the tissue microarray cores per patient sample. The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


B ) Mean ( B ) pIRAK4 staining intensity in CLL - infiltrated ( n = 100 for pIRAK4 ) and tumour - free lymph node biopsies n = 100 for pIRAK4 ) after background subtraction ( y axis ) , p - values from Student ' s t - test . Each dot represents the mean of all cells in the tissue microarray cores per patient sample . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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B) Mean (B) pIRAK4 staining intensity in CLL-infiltrated (n=100 for pIRAK4) and tumour-free lymph node biopsies n=100 for pIRAK4) after background subtraction (y axis), p-values from Student's t-test. Each dot represents the mean of all cells in the tissue microarray cores per patient sample. The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


Example images of immunohistochemistry sections : ( C + D ) pSTAT6 levels in ( C ) CLL - infiltrated and ( D ) tumour - free samples . Scale bar defines 20µm in all images .

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Example images of immunohistochemistry sections: (C + D) pSTAT6 levels in (C) CLL-infiltrated and (D) tumour-free samples. Scale bar defines 20µm in all images.


Example images of immunohistochemistry sections : ( E + F ) pIRAK4 levels in ( E ) CLL - infiltrated and ( F ) tumour - free samples . Scale bar defines 20µm in all images .

Example images of immunohistochemistry sections: (E + F) pIRAK4 levels in (E) CLL-infiltrated and (F) tumour-free samples. Scale bar defines 20µm in all images.


ISRE - luciferase reporter assay in 293T cells infected with BTV - 8 or mock - infected ( Control ) and treated with 1000 U / ml of universal - IFN Luminescence fold induction was measured at different time points p . i . Mean luciferase activity fold inductions in control or BTV - infected in three experiments are presented . *

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ISRE-luciferase reporter assay in 293T cells infected with BTV-8 or mock-infected (Control) and treated with 1000 U/ml of universal-IFN Luminescence fold induction was measured at different time points p.i. Mean luciferase activity fold inductions in control or BTV-infected in three experiments are presented. *


GAS - luciferase reporter assay in Vero cells infected with BTV - 8 or mock - infected ( Control ) and treated wit 5 ng / ml of IFN - γ . Luminescence fold induction was measured at different time points p . i . Mean luciferase activity fold inductions in control or BTV - infected in three experiments are

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GAS-luciferase reporter assay in Vero cells infected with BTV-8 or mock-infected (Control) and treated wit 5 ng/ml of IFN-γ. Luminescence fold induction was measured at different time points p.i. Mean luciferase activity fold inductions in control or BTV-infected in three experiments are presented


qRT - PCR analysis of ISGs induction in mock - or BTV - 8 - infected 293T cells after type I IFN treatment for 8 hr . Mean mRNA fold inductions for 3 experiments are presented .

qRT-PCR analysis of ISGs induction in mock- or BTV-8-infected 293T cells after type I IFN treatment for 8 hr. Mean mRNA fold inductions for 3 experiments are presented.


qRT - PCR analysis of ISGs induction in mock - or BTV - 8 - infected Vero cells after type II IFN treatment for 8 hr . Mean mRNA fold inductions for 3 experiments are presented .

qRT-PCR analysis of ISGs induction in mock- or BTV-8-infected Vero cells after type II IFN treatment for 8 hr. Mean mRNA fold inductions for 3 experiments are presented.


Immunoblot of Vero cells infected with BTV - 8 for 16 h , and probed for total and phosphorylated STAT1 / 2 levels .

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Immunoblot of Vero cells infected with BTV-8 for 16 h, and probed for total and phosphorylated STAT1/2 levels.


Vero cells infected with BTV - 8 were fixed at 16hpi , and stained with anti - BTV - VP7 antibody .

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Vero cells infected with BTV-8 were fixed at 16hpi, and stained with anti-BTV-VP7 antibody.


STAT1 phosphorylation in IFN - I - treated Vero cells infected with BTV - 8 detected by ( C ) flow cytometry and ( D ) confocal microscopy ( Scale bar = 30 µm ) . Means ± SD of P - STAT1 mean fluorescence intensity ( MFI ) in 3 experiments are shown .

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STAT1 phosphorylation in IFN-I-treated Vero cells infected with BTV-8 detected by (C) flow cytometry and (D) confocal microscopy (Scale bar = 30 µm). Means ± SD of P-STAT1 mean fluorescence intensity (MFI) in 3 experiments are shown.


STAT2 expression in BTV - 8 - infected Vero cells detected by ( E ) flow cytometry and ( F ) confocal microscopy ( Scale bar = 30 µm ) . Means ± SD of STAT2 MFI in 3 experiments are shown .

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STAT2 expression in BTV-8-infected Vero cells detected by (E) flow cytometry and (F) confocal microscopy (Scale bar = 30 µm). Means ± SD of STAT2 MFI in 3 experiments are shown.


Vero cells were infected at MOI 1 ( or mock infected ) and treated with proteasome inhibitors MG132 ( MG 20 µM ) , lactacystin ( LC 20 µM ) , chloroquine ( CQ 50 µM ) , 3 - MA ( 5 mM ) , or bafilomycin - A1 ( BAF - A1 0 . 2 µM ) for the final 6h . Representative flow cytometry histogram of BTV - VP7 expression in inhibitor - treated cells .

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Vero cells were infected at MOI 1 (or mock infected) and treated with proteasome inhibitors MG132 (MG 20 µM), lactacystin (LC 20 µM), chloroquine (CQ 50 µM), 3-MA (5 mM), or bafilomycin-A1 (BAF-A1 0.2 µM) for the final 6h. Representative flow cytometry histogram of BTV-VP7 expression in inhibitor-treated cells.


Vero cells were infected at MOI 1 ( or mock infected ) and treated with proteasome inhibitors MG132 ( MG 20 µM ) , lactacystin ( LC 20 µM ) , chloroquine ( CQ 50 µM ) , 3 - MA ( 5 mM ) , for the final 6h . Immunoblots probed for STAT1 , STAT2 , LC3 and GAPDH expression .

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Vero cells were infected at MOI 1 (or mock infected) and treated with proteasome inhibitors MG132 (MG 20 µM), lactacystin (LC 20 µM), chloroquine (CQ 50 µM), 3-MA (5 mM), for the final 6h. Immunoblots probed for STAT1, STAT2, LC3 and GAPDH expression.


Vero cells were infected at MOI 1 ( or mock infected ) and treated with bafilomycin - A1 ( BAF - A1 0 . 2 µM ) for the final 6h . Immunoblots probed for STAT1 , STAT2 , LC3 and GAPDH expression .

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Vero cells were infected at MOI 1 (or mock infected) and treated with bafilomycin-A1 (BAF-A1 0.2 µM) for the final 6h. Immunoblots probed for STAT1, STAT2, LC3 and GAPDH expression.


Immunofluorescence of BTV - 8 or mock - infected Vero cells ( treated for the last 6h with BAF - A1 ) stained for BTV proteins , STAT2 and p62 . Inset shows detail of STAT2 and p62 colocalization . Nuclei were counterstained with DAPI . Arrowheads in inset indicate p62 and STAT2 colocalization . Scale bar = 20 μm . Inset scale bar = 3 μm .

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Immunofluorescence of BTV-8 or mock-infected Vero cells (treated for the last 6h with BAF-A1) stained for BTV proteins, STAT2 and p62. Inset shows detail of STAT2 and p62 colocalization. Nuclei were counterstained with DAPI. Arrowheads in inset indicate p62 and STAT2 colocalization. Scale bar = 20 μm. Inset scale bar = 3 μm.


ISRE - luciferase reporter assays in 293T cells stimulated with IFN - U co - transfected with BTV - VP6 , - VP7 , - NS1 , - NS2 , - NS3 , or - NS4 - expressing plasmids or empty expression plasmid ( Control ) . Representative mean fold inductions of luciferase activity of three independent experiments are shown .

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ISRE-luciferase reporter assays in 293T cells stimulated with IFN-U co-transfected with BTV -VP6, -VP7, -NS1, -NS2, -NS3, or -NS4-expressing plasmids or empty expression plasmid (Control). Representative mean fold inductions of luciferase activity of three independent experiments are shown.


GAS - luciferase reporter assays in Vero cells stimulated with IFN - γ co - transfected with BTV - VP6 , - VP7 , - NS1 , - NS2 , - NS3 , or - NS4 - expressing plasmids or empty expression plasmid ( Control ) . Representative mean fold inductions of luciferase activity of three independent experiments are shown .

GAS-luciferase reporter assays in Vero cells stimulated with IFN-γ co-transfected with BTV -VP6, -VP7, -NS1, -NS2, -NS3, or -NS4-expressing plasmids or empty expression plasmid (Control). Representative mean fold inductions of luciferase activity of three independent experiments are shown.


Immunoblot of 293T cells transfected with BTV - NS2 , - NS3 , - NS4 ( FLAG ) plasmids and NiV - V ( HA ) and WNV - NS5 ( HA ) used as control for STAT1 phosphorylation inhibition and DENV - NS5 ( HA ) used as control for STAT2 degradation .

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Immunoblot of 293T cells transfected with BTV -NS2, -NS3, -NS4 (FLAG) plasmids and NiV-V (HA) and WNV-NS5 (HA) used as control for STAT1 phosphorylation inhibition and DENV-NS5 (HA) used as control for STAT2 degradation.


Three independent immunoblot experiments as in ( C ) were quantified for STAT1 , P - STAT1 , STAT2 and P - STAT2 . Mean ± SD normalized to GAPDH and non - transfected cells .

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Three independent immunoblot experiments as in (C) were quantified for STAT1, P-STAT1, STAT2 and P-STAT2. Mean ± SD normalized to GAPDH and non-transfected cells.


Immunoprecipitation ( IP ) for STAT1 / 2 binding using 293T transfected with BTV - NS2 , - NS3 , or - NS4 plasmids . NiV - V used as positive control for STAT1 and STAT2 binding , DENV - NS5 used as positive control for STAT2 binding . Cells were treated ( or left untreated ) with 1000 U / ml universal - IFN for the final 16 h . Immunoblots of WCE and IP were probed ( IB ) for STAT1 , STAT2 , HA ( NiV V , DENV NS5 ) , FLAG ( NS2 , NS3 , NS4 ) and GAPDH .

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Immunoprecipitation (IP) for STAT1/2 binding using 293T transfected with BTV -NS2, -NS3, or -NS4 plasmids. NiV-V used as positive control for STAT1 and STAT2 binding, DENV-NS5 used as positive control for STAT2 binding. Cells were treated (or left untreated) with 1000 U/ml universal-IFN for the final 16 h. Immunoblots of WCE and IP were probed (IB) for STAT1, STAT2, HA (NiV V, DENV NS5), FLAG (NS2, NS3, NS4) and GAPDH.


STAT1 phosphorylation and ( G ) STAT2 nuclear translocation in BTV - NS3 - transfected Vero cells detected by immunofluorescence and confocal microscopy after 30 min IFN treatment . ( Scale bar = 20 µm ) . Arrowheads in merge images indicate examples of transfected cells .

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STAT1 phosphorylation and (G) STAT2 nuclear translocation in BTV-NS3-transfected Vero cells detected by immunofluorescence and confocal microscopy after 30 min IFN treatment. (Scale bar = 20 µm). Arrowheads in merge images indicate examples of transfected cells.


Immunoblots of BTV - NS3 - transfected 293T cells treated with proteasome inhibitors MG or LC . Membranes were probed for STAT1 , STAT2 , BTV - NS3 ( FLAG ) and GAPDH . ( B - D ) Immunoblots of BTV - NS3 - transfected 293T cells treated with increasing concentration of lysosome acidification inhibitors ( B ) CQ or ( C ) NH4Cl or with increasing concentration of the ( D ) autophagosome formation inhibitor 3 - MA , and probed ( IB ) for STAT1 , STAT2 , BTV - NS3 ( FLAG ) and GAPDH expression .

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

Immunoblots of BTV-NS3-transfected 293T cells treated with proteasome inhibitors MG or LC. Membranes were probed for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH. (B-D) Immunoblots of BTV-NS3-transfected 293T cells treated with increasing concentration of lysosome acidification inhibitors (B) CQ or (C) NH4Cl or with increasing concentration of the (D) autophagosome formation inhibitor 3-MA, and probed (IB) for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH expression.


293T cells were co - transfected with NS3 expression plasmid ( or empty plasmid as control ) and siRNA for Atg7 Lysates were obtained from FACS - sorted transfected cells . Immunoblots of sorted cells were probed for STAT1 ; STAT2 ; Atg7 FLAG - tagged NS3 ; LC3 and GAPDH .

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293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Atg7 Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Atg7 FLAG-tagged NS3; LC3 and GAPDH.


293T cells were co - transfected with NS3 expression plasmid ( or empty plasmid as control ) and siRNA for Beclin - 1 ( or siRNA control or no RNA as controls ) . Lysates were obtained from FACS - sorted transfected cells . Immunoblots of sorted cells were probed for STAT1 ; STAT2 ; Beclin - 1 FLAG - tagged NS3 ; LC3 and GAPDH .

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

293T cells were co-transfected with NS3 expression plasmid (or empty plasmid as control) and siRNA for Beclin-1 (or siRNA control or no RNA as controls). Lysates were obtained from FACS-sorted transfected cells. Immunoblots of sorted cells were probed for STAT1; STAT2; Beclin-1 FLAG-tagged NS3; LC3 and GAPDH.


Representative flow cytometry dot plots of Vero cells infected with rgBTV - 8 or BTV - 8 ΔNS3 and stained for BTV - VP7 . Gating on VP7 + cells was used for sorting to obtain lysates from infected cells .

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Representative flow cytometry dot plots of Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and stained for BTV-VP7. Gating on VP7+ cells was used for sorting to obtain lysates from infected cells.


Immunoblot of FACS - sorted Vero cells infected with rgBTV - 8 or BTV - 8 ΔNS3 and treated with IFN - U for 30min prior to fixation , staining and sorting . Membranes were probed for STAT1 , P - STAT1 , STAT2 and GAPDH .

Immunoblot of FACS-sorted Vero cells infected with rgBTV-8 or BTV-8 ΔNS3 and treated with IFN-U for 30min prior to fixation, staining and sorting. Membranes were probed for STAT1, P-STAT1, STAT2 and GAPDH.


Immunofluorescence confocal images of Vero cells infected with rgBTV - 8 , BTV - 8 ΔNS3 or mock - infected and treated with IFN - U for 30min prior to fixation and staining . Cells were stained for BTV - VP7 and ( I ) P - STAT1 or ( J ) STAT2 . Nuclei were counterstained with DAPI . ( Scale bar = 30 μm ) . ( I ) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV - 8 ΔNS3 infected cells . ( J ) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV - 8 ΔNS3 - infected cells .

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Immunofluorescence confocal images of Vero cells infected with rgBTV-8, BTV-8 ΔNS3 or mock-infected and treated with IFN-U for 30min prior to fixation and staining. Cells were stained for BTV-VP7 and (I) P-STAT1 or (J) STAT2. Nuclei were counterstained with DAPI. (Scale bar = 30 μm). (I) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV-8 ΔNS3 infected cells. (J) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV-8 ΔNS3-infected cells.


WCE and FLAG - tag NS3 IP assay immunoblots of 293T transfected with BTV - NS3 - FLAG plasmids and increasing amounts of Ubiquitin - HA plasmids , and probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 ( FLAG ) or GAPDH expression .

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WCE and FLAG-tag NS3 IP assay immunoblots of 293T transfected with BTV-NS3-FLAG plasmids and increasing amounts of Ubiquitin-HA plasmids, and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG) or GAPDH expression.


Immunofluorescence staining of Vero cells co - transfected with BTV - NS3 and ubiquitin and acquired with Airyscan super - resolution . Scale bar = 10 μm . Inset shows detailed area and arrowheads indicate NS3 and ubiquitin signal co - incidence ( scale bar = 2 μm ) .

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Immunofluorescence staining of Vero cells co-transfected with BTV-NS3 and ubiquitin and acquired with Airyscan super-resolution. Scale bar = 10 μm. Inset shows detailed area and arrowheads indicate NS3 and ubiquitin signal co-incidence (scale bar = 2 μm).


Fluorescence intensity profile of cell section indicated by the white line ( blue : DAPI , green : NS3 ; red : Ub ) . Arrowheads on image and arrows on intensity profile indicate matching fluorescence signal for NS3 and Ub .

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Fluorescence intensity profile of cell section indicated by the white line (blue: DAPI, green: NS3; red: Ub). Arrowheads on image and arrows on intensity profile indicate matching fluorescence signal for NS3 and Ub.


FLAG - tag IP assay of BTV - NS3 and WCE immunoblot of 293T co - transfected with BTV FLAG - NS3 - , - NS3 - K13R , - NS3 - K15R , - NS3 - K13 / 15R or - NS3 - PPRY / AARH plasmid and Ubiquitin - HA plasmid and probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 ( FLAG ) , STAT2 and GAPDH expression .

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

FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected with BTV FLAG-NS3-, -NS3-K13R, -NS3-K15R, -NS3-K13/15R or -NS3-PPRY/AARH plasmid and Ubiquitin-HA plasmid and probed (IB) for ubiquitin (HA), BTV-NS3 (FLAG), STAT2 and GAPDH expression.


WCE and HA - tag Ubiquitin IP assay immunoblots of 293T transfected with Ubiquitin - HA plasmids and increasing amounts of BTV - FLAG - NS3 - K13 / 15R , and probed ( IB ) for ubiquitin ( HA ) , BTV - NS3 - K13 / 15R ( FLAG ) or GAPDH expression .

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WCE and HA-tag Ubiquitin IP assay immunoblots of 293T transfected with Ubiquitin-HA plasmids and increasing amounts of BTV-FLAG-NS3-K13/15R, and probed (IB) for ubiquitin (HA), BTV-NS3-K13/15R (FLAG) or GAPDH expression.


ISRE - driven luciferase reporter assay in 293T cells transfected with empty expression plasmid ( Control ) , BTV - NS2 ( 300 ng ) , - NS3 ( 50 , 300 and 600 ng ) , or - NS3 - K13 / 15R ( 50 , 300 and 600 ng ) - expressing plasmids . Mean % inductions of luciferase activity of three independent experiments are shown .

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ISRE-driven luciferase reporter assay in 293T cells transfected with empty expression plasmid (Control), BTV -NS2 (300 ng), -NS3 (50, 300 and 600 ng), or -NS3-K13/15R (50, 300 and 600 ng)-expressing plasmids. Mean % inductions of luciferase activity of three independent experiments are shown.


FLAG - tag IP assay of BTV - NS3 and WCE immunoblot of 293T co - transfected FLAG - BTV - NS3 plasmid and HA - UB - WT or HA - UB lysine ( K to R ) mutants . IP were probed ( IB ) for ubiquitin ( HA ) and BTV - NS3 ( FLAG ) expression . WCE were probed for STAT1 , STAT2 , ubiquitin ( HA ) , BTV - NS3 ( FLAG ) , and GAPDH expression .

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FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected FLAG-BTV-NS3 plasmid and HA-UB-WT or HA-UB lysine (K to R) mutants. IP were probed (IB) for ubiquitin (HA) and BTV-NS3 (FLAG) expression. WCE were probed for STAT1, STAT2, ubiquitin (HA), BTV-NS3 (FLAG), and GAPDH expression.


( C - D ) RNA - Seq data from GTEx of the normalized expression of human CSAG1 ( C ) and CSAG2 ( D ) in the indicated tissues . Number of biological replicates are as follows : testis 361 , brain 255 , small intestine 187 , white adipose 663 , muscle 803 , skin 604 , esophagus 555 , stomach 359 , liver 226 , heart 429 . blood vessel 663 , lung 578 , colon 373 , nerve 619 , pituitary 283 , blood 755 , adrenal gland 258 , kidney 85 , prostate 245 , salivary gland 162 , ovary 180 , breast 459 , pancreas 328 , vagina 156 , uterus 142 , spleen 241 , fallopian tube 9 , bladder 21 , cervix 10 . Central band indicates median , boxes define 25 and 75 percentiles , and whiskers define 5 and 95 percentiles .

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(C-D) RNA-Seq data from GTEx of the normalized expression of human CSAG1 (C) and CSAG2 (D) in the indicated tissues. Number of biological replicates are as follows: testis 361, brain 255, small intestine 187, white adipose 663, muscle 803, skin 604, esophagus 555, stomach 359, liver 226, heart 429. blood vessel 663, lung 578, colon 373, nerve 619, pituitary 283, blood 755, adrenal gland 258, kidney 85, prostate 245, salivary gland 162, ovary 180, breast 459, pancreas 328, vagina 156, uterus 142, spleen 241, fallopian tube 9, bladder 21, cervix 10. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.


( E - F ) Expression of CSAG1 ( E ) and CSAG2 ( F ) in normal adjacent ( blue ) or tumor tissue ( red ) from TCGA RNA - Seq data . N . D . represents normal adjacent is not available . Data visualized by firebrowse . Number of biological replicates are as follows : ACC 41 , BLCA 307 , BRCA 524 , CESC 175 , CHOL 11 , COAD 153 , COADREAD 232 , DLBC 36 , ESCA 121 , HNSC 416 , KICH 46 , KIPAN 515 , KIRC 411 , KIRP 58 , LAML 7 , LGG 528 , LUAD 301 , LUSC 400 , MESO 31 , OV 183 , PCPG 70 , PRAD 204 , READ 79 , SARC 156 , SKCM 422 , STAD 288 , STES 409 , TGCT 126 , THCA 408 , THYM 75 , UCEC 306 , and UCS 37 . Central band indicates median , boxes define 25 and 75 percentiles , and whiskers define 5 and 95 percentiles .

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(E-F) Expression of CSAG1 (E) and CSAG2 (F) in normal adjacent (blue) or tumor tissue (red) from TCGA RNA-Seq data. N.D. represents normal adjacent is not available. Data visualized by firebrowse. Number of biological replicates are as follows: ACC 41, BLCA 307, BRCA 524, CESC 175, CHOL 11, COAD 153, COADREAD 232, DLBC 36, ESCA 121, HNSC 416, KICH 46, KIPAN 515, KIRC 411, KIRP 58, LAML 7, LGG 528, LUAD 301, LUSC 400, MESO 31, OV 183, PCPG 70, PRAD 204, READ 79, SARC 156, SKCM 422, STAD 288, STES 409, TGCT 126, THCA 408, THYM 75, UCEC 306, and UCS 37. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.


( G ) Xq28 MAGE and CSAG genes are frequently co - expressed in lung squamous carcinoma tumors ( n = 184 ) . RNA - Seq gene expression data analyzed from TCGA . Statistics shown were calculated by linear regression analysis .

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(G) Xq28 MAGE and CSAG genes are frequently co-expressed in lung squamous carcinoma tumors (n=184). RNA-Seq gene expression data analyzed from TCGA. Statistics shown were calculated by linear regression analysis.


( H - K ) Expression of CSAG1 ( H - I ) or CSAG2 ( J - K ) in patients from indicated cancer types correlates with poor overall survival . Data is from TCGA and visualized by kmplotter .

(H-K) Expression of CSAG1 (H-I) or CSAG2 (J-K) in patients from indicated cancer types correlates with poor overall survival. Data is from TCGA and visualized by kmplotter.


( A - B ) The effect of stable knockdown CSAG2 in HCT116 ( A ) or A375 ( B ) were assayed for clonogenic growth on plastic for 7 days . The number of colonies was quantified and is shown . Data are represented as the mean ± SD , n = 3 biological replicates .

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(A-B) The effect of stable knockdown CSAG2 in HCT116 (A) or A375 (B) were assayed for clonogenic growth on plastic for 7 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.


( C - D ) The effect of stable knockdown CSAG2 in HCT116 ( C ) or A375 ( D ) were determined for anchorage - independent growth in soft agar colony formation assays for 14 days . The number of colonies was quantified and is shown . Data are represented as the mean ± SD , n = 3 biological replicates .

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(C-D) The effect of stable knockdown CSAG2 in HCT116 (C) or A375 (D) were determined for anchorage-independent growth in soft agar colony formation assays for 14 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.


( E - F ) Knockdown of CSAG2 in A375 ( F ) or HCT116 ( E ) cells decreases xenograft tumor growth in mice . Cells with doxycycline - inducible shRNAs were implanted into NOD SCID gamma mice . Doxycycline ( 2 mg / mL ) was administered via drinking water . Tumor growth was monitored over time . Data are represented as the mean ± SD , n = 6 mice for each group .

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

(E-F) Knockdown of CSAG2 in A375 (F) or HCT116 (E) cells decreases xenograft tumor growth in mice. Cells with doxycycline-inducible shRNAs were implanted into NOD SCID gamma mice. Doxycycline (2 mg/mL) was administered via drinking water. Tumor growth was monitored over time. Data are represented as the mean ± SD, n=6 mice for each group.


( G - H ) Stable expression of CSAG2 in HCT116 ( G ) and CSAG2 - negative H460 ( H ) cells increases anchorage - independent growth in soft agar colony formation assays , n = 3 biological replicates . Data are represented as the mean ± SD .

(G-H) Stable expression of CSAG2 in HCT116 (G) and CSAG2-negative H460 (H) cells increases anchorage-independent growth in soft agar colony formation assays, n=3 biological replicates. Data are represented as the mean ± SD.


( A ) HEK293 cells stably expressing TAP - vector , TAP - CSAG1 , or TAP - CSAG2 were subjected to pull - down followed by SDS - PAGE and LC - MS / MS . Spectral counts ( SC ) of the identified proteins in TAP - CSAG1 and TAP - CSAG2 pulldowns , but absence in TAP - vector control , are shown .

(A) HEK293 cells stably expressing TAP-vector, TAP-CSAG1, or TAP-CSAG2 were subjected to pull-down followed by SDS-PAGE and LC-MS/MS. Spectral counts (SC) of the identified proteins in TAP-CSAG1 and TAP-CSAG2 pulldowns, but absence in TAP-vector control, are shown.


( B ) HEK293FT cells stably expressing HA - SIRT1 were transfected with Myc - CSAG1 or Myc - CSAG2 for 48 h before IP with anti - HA followed by SDS - PAGE and immunoblotting for indicated proteins .

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(B) HEK293FT cells stably expressing HA-SIRT1 were transfected with Myc-CSAG1 or Myc-CSAG2 for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for indicated proteins.


( C ) Recombinant GST - SIRT1 , but not GST , binds in vitro translated Myc - CSAG2 . Myc - tagged CSAG2 was in vitro translated and GST - pulldown experiments were performed with recombinant GST or GST - SIRT1 followed by SDS - PAGE and immunoblotting for anti - Myc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Recombinant GST-SIRT1, but not GST, binds in vitro translated Myc-CSAG2. Myc-tagged CSAG2 was in vitro translated and GST-pulldown experiments were performed with recombinant GST or GST-SIRT1 followed by SDS-PAGE and immunoblotting for anti-Myc.


Expression of CSAG2 decreased p53 ac - K382 levels and downregulated the expression of downstream p53 target genes in H460 cells . Cells were treated by either 1 μM doxorubicin ( D - E ) with 0 . 4 μM TSA for the indicated times . Cells were then harvested and blotted for the indicated proteins . Quantitation of expression levels is shown ( E Ac - p53 K382 levels normalized to total p53 , whereas all other proteins were normalized to GAPDH .

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

Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by either 1 μM doxorubicin (D-E) with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown (E Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.


Expression of CSAG2 decreased p53 ac - K382 levels and downregulated the expression of downstream p53 target genes in H460 cells . Cells were treated by 20 μM etoposide with 0 . 4 μM TSA for the indicated times . Ac - p53 K382 levels normalized to total p53 , whereas all other proteins were normalized to GAPDH .

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

Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by 20 μM etoposide with 0.4 μM TSA for the indicated times. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.


Expression of CSAG2 decreased p53 ac - K382 levels and downregulated the expression of downstream p53 target genes in H460 cells . Cells were treated by 20 μM etoposide with 0 . 4 μM TSA for the indicated times . Cells were then harvested and blotted for the indicated proteins . Quantitation of expression levels is shown G ) as the mean ± SD , n = 3 biological replicates . Ac - p53 K382 levels normalized to total p53 , whereas all other proteins were normalized to GAPDH .

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

Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by 20 μM etoposide with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown G) as the mean ± SD, n=3 biological replicates. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.


Knockdown of endogenous CSAG2 in HCT116 ( H - I ) cells increased p53 ac - K382 levels . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cells were then harvested and blotted for the indicated proteins . Quantitation of ac - p53 K382 levels relative to total p53 is shown ( I as the mean ± SD , n = 3 biological replicates .

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

Knockdown of endogenous CSAG2 in HCT116 (H-I) cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown (I as the mean ± SD, n=3 biological replicates.


Knockdown of endogenous CSAG2 in A375 cells increased p53 ac - K382 levels . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cells were then harvested and blotted for the indicated proteins .

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

Knockdown of endogenous CSAG2 in A375 cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins.


Knockdown of endogenous CSAG2 in A375 cells increased p53 ac - K382 levels . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cells were then harvested and blotted for the indicated proteins . Quantitation of ac - p53 K382 levels relative to total p53 is shown K ) as the mean ± SD , n = 3 biological replicates .

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

Knockdown of endogenous CSAG2 in A375 cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown K) as the mean ± SD, n=3 biological replicates.


( L - M ) Re - expression of CSAG2 rescued p53 ac - K382 levels in CSAG2 knockdown stable cell lines . Myc - CSAG2 was transfected in HCT116 ( L ) or A375 ( M ) CSAG2 knockdown stable cell lines . Cells were treated with 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr and then blotted for the indicated proteins . Quantitation of ac - p53 K382 levels relative to total p53 is shown as the mean ± SD , n = 3 biological replicates .

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(L-M) Re-expression of CSAG2 rescued p53 ac-K382 levels in CSAG2 knockdown stable cell lines. Myc-CSAG2 was transfected in HCT116 (L) or A375 (M) CSAG2 knockdown stable cell lines. Cells were treated with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr and then blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown as the mean ± SD, n=3 biological replicates.


( N ) CSAG2 does not enhance anchorage - independent growth of HCT116 p53 - null cells . CSAG2 was stably expressed in isogenic HCT116 p53 - wild - type or p53 - null cells . Anchorage - independent growth in soft agar colony formation assays was determined after 14 days . The number of colonies was quantified , normalized to each vector control cell line , and is shown as the mean ± SD , n = 3 biological replicates .

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(N) CSAG2 does not enhance anchorage-independent growth of HCT116 p53-null cells. CSAG2 was stably expressed in isogenic HCT116 p53-wild-type or p53-null cells. Anchorage-independent growth in soft agar colony formation assays was determined after 14 days. The number of colonies was quantified, normalized to each vector control cell line, and is shown as the mean ± SD, n=3 biological replicates.


( A ) Knockdown of SIRT1 abolished CSAG2 - mediated decrease in ac - p53 K382 . CSAG2 over - expressing HCT116 were transfected with SIRT1 siRNA for 72 hr before doxorubicin / TSA treatment for 6 hr . Cell lysates were blotted for the indicated proteins .

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(A) Knockdown of SIRT1 abolished CSAG2-mediated decrease in ac-p53 K382. CSAG2 over-expressing HCT116 were transfected with SIRT1 siRNA for 72 hr before doxorubicin/TSA treatment for 6 hr. Cell lysates were blotted for the indicated proteins.


( B ) Inhibition of SIRT1 enzymatic activity blocked CSAG2 - induced decrease in p53 ac - K382 . CSAG2 over - expressing HCT116 cells were treated with 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . Cell lysates were blotted for the indicated proteins .

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(B) Inhibition of SIRT1 enzymatic activity blocked CSAG2-induced decrease in p53 ac-K382. CSAG2 over-expressing HCT116 cells were treated with 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cell lysates were blotted for the indicated proteins.


Inhibition of SIRT1 enzymatic activity prevents CSAG2 - knockdown increase in p53 ac - K382 . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . Cells were then harvested and blotted for the indicated proteins ( C )

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Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins (C)


Inhibition of SIRT1 enzymatic activity prevents CSAG2 - knockdown increase in p53 ac - K382 . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . Cells were then harvested and blotted for the indicated proteins and quantitated ( D ; n = 3 biological replicates ) . Data shown as mean ± SD .

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Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins and quantitated (D; n=3 biological replicates). Data shown as mean ± SD.


( E ) CSAG2 binds catalytically inactive SIRT1 H363Y . Cells were transfected with the indicated constructs before anti - Myc IP and blotting for the indicated proteins .

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(E) CSAG2 binds catalytically inactive SIRT1 H363Y. Cells were transfected with the indicated constructs before anti-Myc IP and blotting for the indicated proteins.


( F ) SIRT1 H363Y blocks CSAG2 regulation of p53 ac - K382 levels . HCT116 cells were transfected with the indicated constructs and blotted for the indicated proteins .

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(F) SIRT1 H363Y blocks CSAG2 regulation of p53 ac-K382 levels. HCT116 cells were transfected with the indicated constructs and blotted for the indicated proteins.


( G ) Inhibition of SIRT1 activity abolished CSAG2 - induced anchorage - independent growth . Soft agar colony formation assays were performed in control or CSAG2 over - expressing HCT116 cells with or without EX - 527 treatment . Data are represented as the mean ± SD , n = 3 biological replicates .

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(G) Inhibition of SIRT1 activity abolished CSAG2-induced anchorage-independent growth. Soft agar colony formation assays were performed in control or CSAG2 over-expressing HCT116 cells with or without EX-527 treatment. Data are represented as the mean ± SD, n=3 biological replicates.


( H ) Summary of region on CSAG2 required for SIRT1 interaction . HEK293FT cells stably expressing HA - SIRT1 were transfected with indicated CSAG2 constructs for 48 h before IP with anti - HA followed by SDS - PAGE and immunoblotting for anti - Myc .

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(H) Summary of region on CSAG2 required for SIRT1 interaction. HEK293FT cells stably expressing HA-SIRT1 were transfected with indicated CSAG2 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.


( I ) CSAG2 Δ37 mutant fails to interact with SIRT1 in vitro . Myc - tagged CSAG2 38 - 127 fragment was in vitro translated and followed by in vitro binding assay with recombinant GST - SIRT1 , SDS - PAGE , and immunoblotting for anti - Myc .

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(I) CSAG2 Δ37 mutant fails to interact with SIRT1 in vitro. Myc-tagged CSAG2 38-127 fragment was in vitro translated and followed by in vitro binding assay with recombinant GST-SIRT1, SDS-PAGE, and immunoblotting for anti-Myc.


( J ) CSAG2 Δ37 mutant does not regulate p53 ac - K382 levels . Cells were transfected with the indicated Myc - CSAG2 constructs for 48 hr before being treated with 1 μM doxorubicin / 0 . 4 μM TSA . Cell lysates were blotted for the indicated proteins .

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(J) CSAG2 Δ37 mutant does not regulate p53 ac-K382 levels. Cells were transfected with the indicated Myc-CSAG2 constructs for 48 hr before being treated with 1 μM doxorubicin/ 0.4 μM TSA. Cell lysates were blotted for the indicated proteins.


( K ) CSAG2 Δ37 fails to promote anchorage - independent growth of H460 cells . Soft agar colony formation assays were performed in CSAG2 - negative H460 cells stably expressing CSAG2 wild - type or CSAG2 Δ37 mutant ( n = 3 biological replicates ) . Data are represented as the mean ± SD .

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(K) CSAG2 Δ37 fails to promote anchorage-independent growth of H460 cells. Soft agar colony formation assays were performed in CSAG2-negative H460 cells stably expressing CSAG2 wild-type or CSAG2 Δ37 mutant (n=3 biological replicates). Data are represented as the mean ± SD.


Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin ( A ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin (A) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of H2O2 ( B ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of H2O2 (B) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin ( C ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin (C) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 cells were treated with the indicated concentrations of H2O2 ( D ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of H2O2 (D) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


( E - F ) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cell lysates were collected and blotted for the indicated proteins ( E ) . Quantitation ( n = 3 biological replicates ) of normalized cleaved PARP levels is shown ( F ) .

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(E-F) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cell lysates were collected and blotted for the indicated proteins (E). Quantitation (n=3 biological replicates) of normalized cleaved PARP levels is shown (F).


( G ) H460 cells were transfected with wild type , Δ37 , or Δ63 CSAG2 for 48 hr . Cells were then treated with the indicated concentrations of H2O2 for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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(G) H460 cells were transfected with wild type, Δ37, or Δ63 CSAG2 for 48 hr. Cells were then treated with the indicated concentrations of H2O2 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


( H ) Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress . CSAG2 over - expressing H460 cells were treated with the indicated concentrations of H2O2 with or without 1 μM EX - 527 for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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(H) Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress. CSAG2 over-expressing H460 cells were treated with the indicated concentrations of H2O2 with or without 1 μM EX-527 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing HCT116 p53 - / - cells were treated with the indicated concentrations of H2O2 ( I ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (I) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing HCT116 p53 - / - cells were treated with the indicated concentrations of doxorubicin ( J ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (J) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 p53 - / - cells were treated with the indicated concentrations of H2O2 ( K ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (K) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 p53 - / - cells were treated with the indicated concentrations of doxorubicin ( L ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (L) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing H1299 ( p53 mutant ) cells were treated with the indicated concentrations of H2O2 ( M ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of H2O2 (M) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing H1299 ( p53 mutant ) cells were treated with the indicated concentrations of doxorubicin ( N ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of doxorubicin (N) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


( A ) CSAG2 does not alter SIRT1 protein levels . HEK293FT cells were transfected with different amounts of Myc - CSAG2 for 48 hr before cells lysates were harvested and immunoblotted for indicated proteins .

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(A) CSAG2 does not alter SIRT1 protein levels. HEK293FT cells were transfected with different amounts of Myc-CSAG2 for 48 hr before cells lysates were harvested and immunoblotted for indicated proteins.


( B ) CSAG2 does not alter SIRT1 subcellular location . U2OS cells were transfected with or without mcherry - CSAG2 for 48 hr before fixation , SIRT1 immunostaining , and imaging . Scale bar : 10 µm .

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(B) CSAG2 does not alter SIRT1 subcellular location. U2OS cells were transfected with or without mcherry-CSAG2 for 48 hr before fixation, SIRT1 immunostaining, and imaging. Scale bar: 10 µm.


( C ) Summary of results mapping interaction region of SIRT1 recognized by CSAG2 . HEK293FT cells stably expressing HA - CSAG2 were transfected with indicated Myc - SIRT1 constructs for 48 h before IP with anti - HA followed by SDS - PAGE and immunoblotting for anti - Myc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Summary of results mapping interaction region of SIRT1 recognized by CSAG2. HEK293FT cells stably expressing HA-CSAG2 were transfected with indicated Myc-SIRT1 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.


( D ) CSAG2 directly binds SIRT1 catalytic domain in vitro . HA - tagged SIRT1 fragments were in vitro translated . In vitro binding assays with recombinant GST or GST - CSAG2 were performed followed by SDS - PAGE and immunoblotting for anti - HA .

(D) CSAG2 directly binds SIRT1 catalytic domain in vitro. HA-tagged SIRT1 fragments were in vitro translated. In vitro binding assays with recombinant GST or GST-CSAG2 were performed followed by SDS-PAGE and immunoblotting for anti-HA.


( E ) CSAG2 enhances SIRT1 activity in vitro . In vitro SIRT1 activity towards p53 ac - K382 peptide was determined in the presence of GST or GST - CSAG2 , n = 3 biological replicates . Nicotinamide ( 1 mM ) was added to the reaction were indicated to block SIRT1 activity .

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(E) CSAG2 enhances SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST or GST-CSAG2, n=3 biological replicates. Nicotinamide (1 mM) was added to the reaction were indicated to block SIRT1 activity.


( F ) CSAG2 Δ37 mutant that does not bind SIRT1 does not enhance SIRT1 activity in vitro . In vitro SIRT1 activity towards p53 ac - K382 peptide was determined in the presence of GST , GST - CSAG2 or GST - CSAG2 38 - 127 , n = 3 biological replicates .

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(F) CSAG2 Δ37 mutant that does not bind SIRT1 does not enhance SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST, GST-CSAG2 or GST-CSAG2 38-127, n=3 biological replicates.


( G ) CSAG2 enhances SIRT1 deacetylation of p53 ac - K382 peptide through increasing kcat , but not Km . not affecting the binding ability with its substrate p53 . SIRT1 activity assays were performed as described above , except varying amounts of p53 ac - K382 fluorometric peptide were used in the presence of saturating NAD + ( 3 mM ) , n = 3 biological replicates .

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(G) CSAG2 enhances SIRT1 deacetylation of p53 ac-K382 peptide through increasing kcat, but not Km. not affecting the binding ability with its substrate p53. SIRT1 activity assays were performed as described above, except varying amounts of p53 ac-K382 fluorometric peptide were used in the presence of saturating NAD+ (3 mM), n=3 biological replicates.


( H - I ) U2OS cells were transfected with Myc - Vector or Myc - CSAG2 for 48 hr before being treated with or without 0 . 4 μM TSA for 6 hr . Cell lysates were blotted for the indicated proteins ( H ) . Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD , n = 3 biological replicates . White circles indicate Myc - Vector and black circles indicate Myc - CSAG2 .

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(H-I) U2OS cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr before being treated with or without 0.4 μM TSA for 6 hr. Cell lysates were blotted for the indicated proteins (H). Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.


( J ) HeLa , U2OS , or HCT116 cells were transfected with Myc - Vector or Myc - CSAG2 for 48 hr . Cell lysates were blotted for the indicated proteins . Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD , n = 3 biological replicates . White circles indicate Myc - Vector and black circles indicate Myc - CSAG2 .

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(J) HeLa, U2OS, or HCT116 cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.


( K ) Knockdown of endogenous CSAG2 in A375 cells increased ac - H4K16 levels . Cell lysates were blotted for the indicated proteins . Quantitation of expression levels of ac - H4K16 relative to total H4 is shown as the mean ± SD , n = 3 biological replicates .

(K) Knockdown of endogenous CSAG2 in A375 cells increased ac-H4K16 levels. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of ac-H4K16 relative to total H4 is shown as the mean ± SD, n=3 biological replicates.


( D , E ) Change in pO2 of osteoclasts in the mice upon exposure to various concentration of oxygen from 21 % to 14 % pO2 . Magnified PLIM images of osteoclasts under different peripheral oxygen saturation ( SpO2 , D ) . The pO2 of each osteoclast in these mice was plotted ( E , n = 47 from three mice for each SpO2 ) . Scale bar , 20 μm .

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(D, E) Change in pO2 of osteoclasts in the mice upon exposure to various concentration of oxygen from 21% to 14% pO2. Magnified PLIM images of osteoclasts under different peripheral oxygen saturation (SpO2, D). The pO2 of each osteoclast in these mice was plotted (E, n = 47 from three mice for each SpO2). Scale bar, 20 μm.


( F ) Effect of physioxia ( 5 % pO2 ) or physiological hypoxia ( 2 % pO2 ) on osteoclastogenesis . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . Scale bar , 100 μm .

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(F) Effect of physioxia (5% pO2) or physiological hypoxia (2% pO2) on osteoclastogenesis. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bar, 100 μm.


( A ) μCT analysis of the femurs of 10 - week - old male mice upon exposure to low oxygen concentration ( 14 % pO2 , n = 5 ) or ambient air ( n = 7 ) , and of 10 - week - old Hif1aRank - / - ; Hif2aRank - / - male mice upon exposure to low oxygen concentration ( 14 % pO2 , n = 6 ) or ambient air ( n = 5 ) for 10 days ( axial view of the metaphyseal region ) . Scale , 0 . 5 mm . ( B ) Histological analysis of the proximal tibias of 10 - week - old male mice upon exposure to low oxygen concentration ( 14 % pO2 ) or ambient air for 10 days , and of 10 - week - old Hif1aRank - / - ; Hif2aRank - / - male mice upon exposure to low oxygen concentration ( 14 % pO2 ) or ambient air ( toluidine blue staining [ arrow , osteoclasts ] ) . Scale , 100 μm . ( C ) Parameters for osteoclasts and osteoblasts during bone morphometric analysis of 10 - week - old male mice under low oxygen concentration ( n = 3 ) or ambient air ( n = 5 ) , and 10 - week - old Hif1aRank - / - ; Hif2aRank - / - male under low oxygen concentration ( n = 3 ) or ambient air ( n = 5 ) for 10 days .

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(A) μCT analysis of the femurs of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2, n = 5) or ambient air (n = 7), and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2, n = 6) or ambient air (n = 5) for 10 days (axial view of the metaphyseal region). Scale, 0.5 mm. (B) Histological analysis of the proximal tibias of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2) or ambient air for 10 days, and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2 ) or ambient air (toluidine blue staining [arrow, osteoclasts]). Scale, 100 μm. (C) Parameters for osteoclasts and osteoblasts during bone morphometric analysis of 10-week-old male mice under low oxygen concentration (n = 3) or ambient air (n = 5), and 10-week-old Hif1aRank-/-; Hif2aRank-/- male under low oxygen concentration (n = 3) or ambient air (n = 5) for 10 days.


( A ) Protein expression of HIF - 1α , HIF - 2α , Blimp1 , NFATc1 , Ctsk and TRAP in control and Hif1aRank - / - ; Hif2aRank - / - BMMs stimulated with RANKL and cultured under different conditions of oxygen concentration for 2 days .

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(A) Protein expression of HIF-1α, HIF-2α, Blimp1, NFATc1, Ctsk and TRAP in control and Hif1aRank-/-; Hif2aRank-/- BMMs stimulated with RANKL and cultured under different conditions of oxygen concentration for 2 days.


( B ) Effect of physioxia ( 5 % pO2 ) or physiological hypoxia ( 2 % pO2 ) on the formation of osteoclast from control and Hif1aRank - / - ; Hif2aRank - / - BMMs . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . Scale bar , 100 μm .

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(B) Effect of physioxia (5% pO2) or physiological hypoxia (2% pO2) on the formation of osteoclast from control and Hif1aRank-/-; Hif2aRank-/- BMMs. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bar, 100 μm.


( C ) Expression of osteoclastogenic and canonical HIF - target genes in wild - type control and Hif1aRank - / - ; Hif2aRank - / - BMMs under conditions of 5 % or 2 % oxygen .

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(C) Expression of osteoclastogenic and canonical HIF-target genes in wild-type control and Hif1aRank-/-; Hif2aRank-/- BMMs under conditions of 5% or 2% oxygen.


( A ) Representative intravital image of calvarial bone marrow of Rosa26GO - ATeam / + ; Tg ( Acp5 - tdTomato ) male mice showing osteoclasts ( left , tdTomato fluorescence ) and FLIM image for ATP changes ( middle and right , fluorescence lifetime of EGFP ) . The FRET efficiencies of each osteoclast were plotted from the mice upon exposure to low oxygen atmosphere ( 14 % pO2 ) or ambient air ( right , n = 11 from three mice for each SpO2 ) .

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(A) Representative intravital image of calvarial bone marrow of Rosa26GO-ATeam/+; Tg(Acp5-tdTomato) male mice showing osteoclasts (left, tdTomato fluorescence) and FLIM image for ATP changes (middle and right, fluorescence lifetime of EGFP). The FRET efficiencies of each osteoclast were plotted from the mice upon exposure to low oxygen atmosphere (14% pO2) or ambient air (right, n = 11 from three mice for each SpO2).


( B ) Representative intravital image of calvarial bone marrow of Tg ( Acp5 - tdTomato ) male mice showing osteoclasts ( left , tdTomato fluorescence ) and NAD ( P ) H autofluorescence lifetime images ( middle and right ) . Average of autofluorescence lifetime of each osteoclast was plotted from the mice upon exposure to low oxygen concentration ( 14 % pO2 ) or ambient air ( right , n = 64 from three mice for each SpO2 ) .

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(B) Representative intravital image of calvarial bone marrow of Tg(Acp5-tdTomato) male mice showing osteoclasts (left, tdTomato fluorescence) and NAD(P)H autofluorescence lifetime images (middle and right). Average of autofluorescence lifetime of each osteoclast was plotted from the mice upon exposure to low oxygen concentration (14% pO2) or ambient air (right, n = 64 from three mice for each SpO2).


( B ) μCT analysis of the femurs of 10 - week - old control ( n = 6 ) , Tet2Rank - / - ( n = 5 ) , Tet3Rank - / - ( n = 5 ) , Tet2Rank - / - ; Tet3aRank + / - ( n = 6 ) , Tet2Rank + / - ; Tet3aRank - / - ( n = 6 ) and Tet2Rank - / - ; Tet3aRank - / - ( n = 4 ) male mice ( axial view of the metaphyseal region ) . ( C ) Histological analysis of the proximal tibias of 10 - week - old control , Tet2Rank - / - , Tet3Rank - / - , Tet2Rank - / - ; Tet3aRank + / - , Tet2Rank + / - ; Tet3aRank - / - and Tet2Rank - / - ; Tet3aRank - / - male mice ( TRAP staining [ red , osteoclasts ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) μCT analysis of the femurs of 10-week-old control (n = 6), Tet2Rank-/- (n = 5), Tet3Rank-/- (n = 5), Tet2Rank-/-; Tet3aRank+/- (n = 6), Tet2Rank+/-; Tet3aRank-/- (n = 6) and Tet2Rank-/-; Tet3aRank-/- (n = 4) male mice (axial view of the metaphyseal region). (C) Histological analysis of the proximal tibias of 10-week-old control, Tet2Rank-/-, Tet3Rank-/-, Tet2Rank-/-; Tet3aRank+/-, Tet2Rank+/-; Tet3aRank-/- and Tet2Rank-/-; Tet3aRank-/- male mice (TRAP staining [red, osteoclasts]).


( E ) Effect of Tet2 and Tet3 double deficiency on osteoclastogenesis . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . ( n = 6 ( control ) , n = 10 ( Tet2Rank - / - ; Tet3aRank + / - ) , n = 3 ( Tet2Rank + / - ; Tet3aRank - / - ) and n = 4 ( Tet2Rank - / - ; Tet3aRank - / - ) biological

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of Tet2 and Tet3 double deficiency on osteoclastogenesis. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). (n = 6 (control), n = 10 (Tet2Rank-/-; Tet3aRank+/-), n = 3 (Tet2Rank+/-; Tet3aRank-/-) and n = 4 (Tet2Rank-/-; Tet3aRank-/-) biological replicates


( B ) MeDIP - qPCR analysis to validate regions that are hypermethylated in Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 2 days . ( C ) Gene expression of TET target candidates in BMMs stimulated with RANKL for 2 days .

(B) MeDIP-qPCR analysis to validate regions that are hypermethylated in Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 2 days. (C) Gene expression of TET target candidates in BMMs stimulated with RANKL for 2 days.


( E ) Bisulfate sequencing analysis of selected region of the second intron of the Prdm1 gene in control and Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 2 days . ( F ) Effect of retroviral NFATc1 expression on osteoclastogenesis of control and Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 3 days . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . ( G ) Bisulfate sequencing analysis of the selected region of the second intron of the Prdm1 gene in BMMs stimulated with RANKL and culture under 5 % and 2 % oxygen for 3 days . ( H ) Effect of retroviral NFATc1 expression on osteoclastogenesis of BMMs stimulated with RANKL and culture under 5 % and 2 % oxygen for 5 days . Control and Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 3 days . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bisulfate sequencing analysis of selected region of the second intron of the Prdm1 gene in control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 2 days. (F) Effect of retroviral NFATc1 expression on osteoclastogenesis of control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). (G) Bisulfate sequencing analysis of the selected region of the second intron of the Prdm1 gene in BMMs stimulated with RANKL and culture under 5% and 2% oxygen for 3 days. (H) Effect of retroviral NFATc1 expression on osteoclastogenesis of BMMs stimulated with RANKL and culture under 5% and 2% oxygen for 5 days. Control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right).


Dual - color SMLM shows co - localization of BAX and DRP1 at the nanoscale . B ) Magnified SMLM images of GFP - BAX and DRP1 assemblies co - localizing up to 30 nm during apoptosis . Scale bar 100 nm . C ) Co - localization control of DRP1 immunostaining with the same primary and different secondary antibodies ( labelled with AF647 and CF680 ) in HeLa cells . Scale bar 100 nm . D ) Quantification of the distance from GFP - BAX to DRP1 structures ( distance measured at the center of each structure ) from dual - color SMLM images . Data are quantified from n = 4 independent experiments with a total of 720 BAX structure .

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

Dual-color SMLM shows co-localization of BAX and DRP1 at the nanoscale. B) Magnified SMLM images of GFP-BAX and DRP1 assemblies co-localizing up to 30 nm during apoptosis. Scale bar 100 nm. C) Co-localization control of DRP1 immunostaining with the same primary and different secondary antibodies (labelled with AF647 and CF680) in HeLa cells. Scale bar 100 nm. D) Quantification of the distance from GFP-BAX to DRP1 structures (distance measured at the center of each structure) from dual-color SMLM images. Data are quantified from n=4 independent experiments with a total of 720 BAX structure.


E , F ) Representative confocal microscopy images of HeLa cells transfected with ddFP RA - BAX and GB - DRP1 ( E ) in untreated or apoptotic ( STS ) conditions , or with RA - BAX and GB - BAX during apoptosis ( F ) . RA - BAX / GB - DRP1 and RA - BAX / GB - BAX complexes shown in green , mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm . Right panels are zoomed areas representing individual and merged channels . Scale bar 5 μm .

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

E,F) Representative confocal microscopy images of HeLa cells transfected with ddFP RA-BAX and GB-DRP1 (E) in untreated or apoptotic (STS) conditions, or with RA-BAX and GB-BAX during apoptosis (F). RA-BAX/GB-DRP1 and RA-BAX/GB-BAX complexes shown in green, mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm. Right panels are zoomed areas representing individual and merged channels. Scale bar 5 μm.


G ) BAX / DRP1 interaction compared to BAX / BAX and BCL ‑ xL / DRP1 by ddFP . % mito - BFP transfected cells that show RA / GB signal was quantified in HeLa cells untreated ( untr . ) and 3 h after apoptosis induction ( STS ) . Box plots represent the interquartile ( box ) , median ( line ) and SD ( whiskers ) of n = 3 independent experiments ( with n = 100 cells each ) . Levels of significance were determined by paired two - tailed Student ´ s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) compared to BAX / DRP1 after apoptotic induction ( STS ) .

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G) BAX/DRP1 interaction compared to BAX/BAX and BCL‑xL/DRP1 by ddFP. % mito-BFP transfected cells that show RA/GB signal was quantified in HeLa cells untreated (untr.) and 3 h after apoptosis induction (STS). Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, *** p < 0.001) compared to BAX/DRP1 after apoptotic induction (STS).


A ) Confocal microscopy time - series images of the increase in cytosolic Smac - GFP ( magenta ) in relation to the detection of RA - BAX / GB - DRP1 complexes ( green ) during apoptosis induction in MEF DRP1 KO cells . Scale bar 10 μm . Images are representative for n = 3 independent experiments .

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

A) Confocal microscopy time-series images of the increase in cytosolic Smac-GFP (magenta) in relation to the detection of RA-BAX/GB-DRP1 complexes (green) during apoptosis induction in MEF DRP1 KO cells. Scale bar 10 μm. Images are representative for n=3 independent experiments.


C ) Quantification of BAX / DRP1 interaction ( % cells with RA - BAX / GB - DRP1 foci normalized to the mito - BFP positive cells , n = 100 cells per experiment ) during inhibition of effector caspases ( Q - VD - OPh ) , the mitochondrial permeability transition pore ( CsA ) versus control ( DMSO ) , with or without apoptosis induction ( + / - STS ) . Box plots represent the interquartile ( box ) , median ( line ) and SD ( whiskers ) . Significance was determined from n = 3 independent experiments ( with n = 100 cells each ) by paired two - tailed Student ´ s t - test ( n . s . indicating p > 0 . 05 ) compared to DMSO control after apoptotic induction ( STS ) .

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

C) Quantification of BAX/DRP1 interaction (% cells with RA-BAX/GB-DRP1 foci normalized to the mito-BFP positive cells, n=100 cells per experiment) during inhibition of effector caspases (Q-VD-OPh), the mitochondrial permeability transition pore (CsA) versus control (DMSO), with or without apoptosis induction (+/- STS). Box plots represent the interquartile (box), median (line) and SD (whiskers). Significance was determined from n=3 independent experiments (with n=100 cells each) by paired two-tailed Student´s t-test (n.s. indicating p > 0.05) compared to DMSO control after apoptotic induction (STS).


C ) Quantification of % CC between DRP1 - AF488 and BAX - AF633 in solution ( grey ) , in the membrane ( violet ) , and in the membrane ( beige ) in presence of excess unlabeled cBID . Box plots represent the interquartile ( outer box ) , mean ( inner box ) , median ( line ) and range ( whiskers ) . Levels of significance were determined by paired two - tailed Student ´ s t - test ( * p < 0 . 05 , * * p < 0 . 01 ) from n = 9 measurements in solution , n = 46 individually measured GUVs in the membrane or n = 17 GUVs in presence of cBID .

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C) Quantification of %CC between DRP1-AF488 and BAX-AF633 in solution (grey), in the membrane (violet), and in the membrane (beige) in presence of excess unlabeled cBID. Box plots represent the interquartile (outer box), mean (inner box), median (line) and range (whiskers). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01) from n=9 measurements in solution, n=46 individually measured GUVs in the membrane or n=17 GUVs in presence of cBID.


E ) Effect of DRP1 on BAX - induced GUV permeabilization . Left : % GUVs permeabilized to Cytochrome c488 ( Cyt c , 12 kDa , beige ) and allophycocianin ( APC , 104 kDa , blue ) in the absence or presence of cBID , BAX and DRP1 combined as indicated . Data are presented as mean ± SD of n = 4 independent experiments . * p < 0 . 05 ( paired two - tailed Student ´ s t - test ) . Right : representative confocal microscopy images showing GUVs ( grey ) in a solution of Cyt c ( green ) and APC ( magenta ) . Scale bar 10 µm .

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

E) Effect of DRP1 on BAX-induced GUV permeabilization. Left: % GUVs permeabilized to Cytochrome c488 (Cyt c, 12 kDa, beige) and allophycocianin (APC, 104 kDa, blue) in the absence or presence of cBID, BAX and DRP1 combined as indicated. Data are presented as mean ± SD of n=4 independent experiments. * p < 0.05 (paired two-tailed Student´s t-test). Right: representative confocal microscopy images showing GUVs (grey) in a solution of Cyt c (green) and APC (magenta). Scale bar 10 µm.


Effect of BAX on DRP1 membrane density and DRP1 - induced shape alterations of liposomes measured by flow cytometry . G ) Membrane density of DRP1 ( corrected fluorescence units , cFU , left graph ) and DRP1 - induced membrane tethering ( indicated by a shape index > 1 , right graph ) in LUVs in the absence or presence of different concentrations of BAX . Data are presented as mean ± SD of n = 3 independent experiments . * p < 0 . 05 ( paired two - tailed Student ´ s t - test ) vs . DRP1 without BAX .

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

Effect of BAX on DRP1 membrane density and DRP1-induced shape alterations of liposomes measured by flow cytometry. G) Membrane density of DRP1 (corrected fluorescence units, cFU, left graph) and DRP1-induced membrane tethering (indicated by a shape index > 1, right graph) in LUVs in the absence or presence of different concentrations of BAX. Data are presented as mean ± SD of n=3 independent experiments. * p < 0.05 (paired two-tailed Student´s t-test) vs. DRP1 without BAX.


BAX peptide array to define interaction - site with DRP1 . D ) Quantification of the relative fluorescence intensity of DRP1 - AF488 at the rim of the GUVs for each BAX peptide of n = 3 individual experiments ( with n = 100 vesicles each ) . Black lines indicate the mean value . Interacting BAX peptides are highlighted in beige .

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BAX peptide array to define interaction-site with DRP1. D) Quantification of the relative fluorescence intensity of DRP1-AF488 at the rim of the GUVs for each BAX peptide of n=3 individual experiments (with n=100 vesicles each). Black lines indicate the mean value. Interacting BAX peptides are highlighted in beige.


B ) Representative confocal microscopy images of U2OS BAX / BAK DKO cells of the interactions between RA - BAX variants and GB - DRP1 , shown as green signal , 3 h after apoptosis induction . Mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm .

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

B) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX variants and GB-DRP1, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.


C - E ) Quantification of the interaction of DRP1 with variants of BAX based on the % cells with detectable RA - BAX / GB - DRP1 fluorescence signal ( interacting cells ) normalized to the number of mito - BFP positive cells . Box plots represent the interquartile ( box ) , median ( line ) and SD ( whiskers ) of n = 3 independent experiments ( with n = 100 cells each ) . Levels of significance were determined by paired two - tailed Student ´ s t - test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) compared to BAX wild type ( WT ) .

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C-E) Quantification of the interaction of DRP1 with variants of BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) compared to BAX wild type (WT).


G ) Representative confocal microscopy images of U2OS BAX / BAK DKO cells of the interactions between RA - BAX and GB - DRP1 variants , shown as green signal , 3 h after apoptosis induction . Mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm .

G) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX and GB-DRP1 variants, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.