Figure 4 ( A ) Coomassie - stained protein gels with cell lysates and concentrated culture supernatants of MCF10A - pIND20 - HPN cells , with ( + DOX ) or without ( - DOX ) hepsin overexpression . M indicates the media only control . R and NR above the gels indicate reducing and non - reducing conditions , respectively . Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry ; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left . The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions , highlighting the part from which fibronectin was identified . Red boxes indicate the lanes that were analyzed by mass specrtometry ( C ) Immunoblot analysis of fibronectin , hepsin , and β - tubulin ( loading control ) in cell lysates from MCF10A - pIND20 - HPN cells with ( DOX + ) or without ( DOX - ) hepsin overexpression . The graph shows the quantification of fibronectin levels . ( D ) Silver - stained protein gel with samples from an in vitro protease activity assay with recombinant hepsin and either full length purified plasma fibronectin ( upper panel ) or the 70 kDa most N - terminal fragment of fibronectin ( lower panel ) . Full - length fibronectin ( 1μg ) or the 70 kDa fibronectin fragment ( 1μg ) were incubated with increasing concentrations of recombinant hepsin . Arrowheads indicate full - length fibronectin , the 70kDa fragment , the 50 kDa cleavage fragment generated by hepsin , and hepsin . The schematic figure shows the full - length fibronectin protein and the 70KDa N - terminal fragment . The red arrow indicates the location of the putative hepsin cleavage site . RGD indicated the RGD - binding domain in fibronectin .

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Figure 4(A) Coomassie-stained protein gels with cell lysates and concentrated culture supernatants of MCF10A-pIND20-HPN cells, with (+DOX) or without (-DOX) hepsin overexpression. M indicates the media only control. R and NR above the gels indicate reducing and non-reducing conditions, respectively. Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left. The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions, highlighting the part from which fibronectin was identified. Red boxes indicate the lanes that were analyzed by mass specrtometry(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) or without (DOX-) hepsin overexpression. The graph shows the quantification of fibronectin levels.(D) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant hepsin and either full length purified plasma fibronectin (upper panel) or the 70 kDa most N-terminal fragment of fibronectin (lower panel). Full-length fibronectin (1μg) or the 70 kDa fibronectin fragment (1μg) were incubated with increasing concentrations of recombinant hepsin. Arrowheads indicate full-length fibronectin, the 70kDa fragment, the 50 kDa cleavage fragment generated by hepsin, and hepsin. The schematic figure shows the full-length fibronectin protein and the 70KDa N-terminal fragment. The red arrow indicates the location of the putative hepsin cleavage site. RGD indicated the RGD-binding domain in fibronectin.


Figure 5 ( A ) Immunoblot analysis of fibronectin in indicated tissues isolated from Wt , Hpn + / - and Hpn - / - mouse ( representative of at least three repeats ) . GAPDH blot is used here as the loading control . ( C ) Immunoblot analysis of fibronectin , hepsin , and β - tubulin ( loading control ) in prostate lysates of control mice or mice with prostate - specific hepsin overexpression N = 2 mice each , the two prostate lobes were run separately ( a and b ) .

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Figure 5(A) Immunoblot analysis of fibronectin in indicated tissues isolated from Wt, Hpn+/- and Hpn-/- mouse (representative of at least three repeats). GAPDH blot is used here as the loading control.(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in prostate lysates of control mice or mice with prostate-specific hepsin overexpression N=2 mice each, the two prostate lobes were run separately (a and b).


Figure 6 ( A ) Immunoblot analysis of fibronectin , pSmad2 / 3 , total Smad2 / 3 , and GAPDH ( loading control ) in cell lysates from MCF10A - pIND20 - HPN cells without hepsin overexpression . The graph depicts quantification of pSmad2 / 3 normalized to total Smad2 / 3 levels , compared to siCtrl ( N = 3 biological repeats ) . ( B ) Immunoblot analysis of cell lysates ( pSmad2 / 3 , total Smad2 / 3 and vinculin as loading control ) and concentrated cell culture supernatant ( anti - V5 for TGFβ ) from MCF10A - pIND20 - HPN pL6 - TGFβ1 - V5 cells without hepsin overexpression . Ponceau staining is shown as a loading control for the western blot with concentrated culture supernatant . The graph depicts quantification of pSmad2 / 3 normalized to total Smad2 / 3 levels , compared to siCtrl ( N = 3 biological repeats ) . ( E ) Immunoblot analysis of fibronectin , pSmad2 / 3 , total Smad2 / 3 , hepsin , and GAPDH ( loading control ) in cell lysates from MCF10A - pIND20 - HPN cells with ( DOX + ) and without ( DOX - ) hepsin overexpression , and with or without fibronectin silencing ( siFN1 ) . The graph depicts quantification of pSmad2 / 3 normalized to total Smad2 / 3 levels ( N = 3 biological repeats ) .

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Figure 6(A) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells without hepsin overexpression. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats). (B) Immunoblot analysis of cell lysates (pSmad2/3, total Smad2/3 and vinculin as loading control) and concentrated cell culture supernatant (anti-V5 for TGFβ) from MCF10A-pIND20-HPN pL6-TGFβ1-V5 cells without hepsin overexpression. Ponceau staining is shown as a loading control for the western blot with concentrated culture supernatant. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats).(E) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, hepsin, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) and without (DOX-) hepsin overexpression, and with or without fibronectin silencing (siFN1). The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels (N = 3 biological repeats).


Figure 1Luciferase - based imaging of drug response in Vegfr3Luc ; Tyr : CreERT2 ; BrafV600E ; Ptenflox / flox mice . Panels labeled as " basal " and " induced " correspond to the bioluminescence of animals prior and 5 weeks after administration of 4OH - tamoxifen ( 5 mM , topical administration , 3 consecutive days ) for the induction of melanomas . ( Right panels ) Treatment with anti - PD - L1 antibody ( αPD - L1 ; clone 10F . 9G2 , 3 weeks ) or the corresponding control IgG ( 200 ug / dose , twice per week , 3 weeks ) ; vemurafenib ( Vem , 50 mg / Kg , oral once per day , 3 weeks ) or BO - 110 ( BO , 0 . 8 mg / kg , twice per week , 3 weeks ) . Scale : p / s / cm2 / sr ( x106 ) . Treatment with BO - 110 of human patient - derived xenografts ( PDX ) implanted in Vegfr3Luc nu / nu . 42 days after implantation ( when systemic luciferase was detected ) , animals were randomized for treatment with vehicle ( V ) or with 0 . 8 mg / kg BO - 110 ( BO , twice per week ) , and luciferase emission was acquired at the indicated times . Scale , p / s / cm2 / sr ( x106 ) .

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Figure 1Luciferase-based imaging of drug response in Vegfr3Luc;Tyr:CreERT2;BrafV600E; Ptenflox/flox mice. Panels labeled as "basal" and "induced" correspond to the bioluminescence of animals prior and 5 weeks after administration of 4OH-tamoxifen (5 mM, topical administration, 3 consecutive days) for the induction of melanomas. (Right panels) Treatment with anti-PD-L1 antibody (αPD-L1; clone 10F.9G2, 3 weeks) or the corresponding control IgG (200 ug/dose, twice per week, 3 weeks); vemurafenib (Vem, 50 mg/Kg, oral once per day, 3 weeks) or BO-110 (BO, 0.8 mg/kg, twice per week, 3 weeks). Scale: p/s/cm2/sr (x106).Treatment with BO-110 of human patient-derived xenografts (PDX) implanted in Vegfr3Luc nu/nu. 42 days after implantation (when systemic luciferase was detected), animals were randomized for treatment with vehicle (V) or with 0.8 mg/kg BO-110 (BO, twice per week), and luciferase emission was acquired at the indicated times. Scale, p/s/cm2/sr (x106).


Figure 2qRT - PCR analysis of relative mRNA levels of MDA5 16 h after treatment of HLEC with 0 . 5 ug / ml BO - 110 ( BO ) , 10 µM vemurafenib ( Vem ) , or vehicle control ( V ) . Data correspond to the mean ± SD of 3 biological replicates . Statistical significance was determined by t - test . qRT - PCR analysis of relative mRNA levels of VEGFR3 16 h after treatment of HLEC with 0 . 5 or 1 ug / ml BO - 110 ( VO ) , 10 µM vemurafenib ( Vem ) , or the corresponding vehicle control ( V ) . Data correspond to the mean ± SD of 3 biological replicates . Statistical significance was determined by ANOVA . Luciferase signal driven by FLT4 ( VEGFR3 ) - promoter transduced into HLEC treated with vehicle ( v ) or BO - 110 ( BO ) at the indicated doses ( ug / ml ) as indicated in methods . Results were normalized to vehicle control . N = 4 biological replicates . Error bars correspond to mean ± SD . Statistical significance was determined by ANOVA . Analysis of apoptotic cells at the indicated time points . HLEC cells were treated with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) for the indicated time points . Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods . Data correspond to the mean ± SD of 3 experiments . Statistical significance was determined by t - test . Relative mRNA levels of VEGFC and VEGFD in the indicated melanoma cell lines 8 h after treatment with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) , as determined by qRT - PCR . Data correspond to the mean ± SD of 3 experiments . Statistical significance was determined by t - test .

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Figure 2qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of HLEC with 0.5 ug/ml BO-110 (BO), 10 µM vemurafenib (Vem), or vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by t-test.   qRT-PCR analysis of relative mRNA levels of VEGFR3 16 h after treatment of HLEC with 0.5 or 1 ug/ml BO-110 (VO), 10 µM vemurafenib (Vem), or the corresponding vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by ANOVA.   Luciferase signal driven by FLT4 (VEGFR3)-promoter transduced into HLEC treated with vehicle (v) or BO-110 (BO) at the indicated doses (ug/ml) as indicated in methods. Results were normalized to vehicle control. N=4 biological replicates. Error bars correspond to mean ± SD. Statistical significance was determined by ANOVA.Analysis of apoptotic cells at the indicated time points. HLEC cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.Relative mRNA levels of VEGFC and VEGFD in the indicated melanoma cell lines 8 h after treatment with vehicle (V) or 0.5 µg/ml BO-110 (BO), as determined by qRT-PCR. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.


Figure 3Inhibitory effect of the indicated doses of BO - 110 ( in µg / ml ) or vehicle ( V ) on MDK mRNA expression determined by qRT - PCR in SK - Mel - 147 ( 16 h after treatment ) . Data correspond to average mRNA levels in three experiments with technical replicates normalized to vehicle control ± SD . Statistical significance was determined by ANOVA . qRT - PCR analysis of relative mRNA levels of MDA5 16 h after treatment of SK - Mel - 147 with the indicated doses of BO - 110 ( in µg / ml ) ( BO ) . Data correspond to the mean ± SD of three experiments with three technical replicates . Statistical significance was determined by ANOVA . Analysis of apoptotic cells at the indicated time points . SK - Mel - 147 cells were treated with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) for the indicated time points . Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods . Data correspond to the mean ± SD of 3 experiments . Statistical significance was determined by t - test . Immunohistochemical analysis of MDK repression ( pink staining ) in SK - Mel - 147 xenografts and PDX lesions after treatment with BO - 110 ( BO , 0 . 8 mg / kg , 2 doses / week ) . Histological staining in tumors extracted from animals treated with vehicle control ( V ) are included as a reference . Nuclei were counterstained in with hematoxylin .

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Figure 3Inhibitory effect of the indicated doses of BO-110 (in µg/ml) or vehicle (V) on MDK mRNA expression determined by qRT-PCR in SK-Mel-147 (16 h after treatment). Data correspond to average mRNA levels in three experiments with technical replicates normalized to vehicle control ± SD. Statistical significance was determined by ANOVA.   qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of SK-Mel-147 with the indicated doses of BO-110 (in µg/ml) (BO). Data correspond to the mean ± SD of three experiments with three technical replicates. Statistical significance was determined by ANOVA.   Analysis of apoptotic cells at the indicated time points. SK-Mel-147 cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.Immunohistochemical analysis of MDK repression (pink staining) in SK-Mel-147 xenografts and PDX lesions after treatment with BO-110 (BO, 0.8 mg/kg, 2 doses/week). Histological staining in tumors extracted from animals treated with vehicle control (V) are included as a reference. Nuclei were counterstained in with hematoxylin.


Figure 4Type I IFN mRNA induction ( IFNA2 and IFNB1 ) in SK - Mel - 147 melanoma cells treated for 16 h with the indicated amounts of BO - 110 ( in mg / ml ) . Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control . Statistical significance was determined by ANOVA . Heatmap depicting expression changes in interferon - related genes ( GO : 0034340 ) in SK - Mel - 147 melanoma cells ( left panel ) and HLEC ( right panel ) treated with vehicle or 0 . 5 g / ml of BO - 110 for 10 hours . CD274 , LAG3 and PDCD1 genes were also included as a reference .

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Figure 4Type I IFN mRNA induction (IFNA2 and IFNB1) in SK-Mel-147 melanoma cells treated for 16 h with the indicated amounts of BO-110 (in mg/ml). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control. Statistical significance was determined by ANOVA.Heatmap depicting expression changes in interferon-related genes (GO:0034340) in SK-Mel-147 melanoma cells (left panel) and HLEC (right panel) treated with vehicle or 0.5 g/ml of BO-110 for 10 hours. CD274, LAG3 and PDCD1 genes were also included as a reference.


Figure 5IFNB1 mRNA induction analyzed by qPCR at the indicated times after BO - 110 treatment ( 0 . 5 µg / ml ) of SK - Mel - 147 melanoma cells or HLEC ( left and right graphs , respectively ) . Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control . Quantification of the impact of BO - 110 as single agent or in the presence of the indicated blocking antibodies for type I interferon ( IFNB1 or IFNAR1 ) . Upper graphs show the effect of these agents on MDK mRNA levels in SK - Mel - 147 melanoma cells . Similar treatments were performed on HLEC for the analysis of VEGFR3 mRNA ( middle graphs ) and tube formation capacity ( lower graphs ) . Data correspond to the mean ± SD of 3 biological replicates in triplicate . Growth of B16 melanoma xenografts in siblings of Ifnar1 + / + , Ifnar1 + / - or Ifnar1 - / - mice . Treatment started 10 days after tumor cell implantation . BO - 110 was administered at 0 . 8 mg / kg , every third day for 2 weeks . N = 6 mice per condition . Graphs show the mean tumor size ± SD at each time point . Statistical significance was determined by Two - Way ANOVA .

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Figure 5IFNB1 mRNA induction analyzed by qPCR at the indicated times after BO-110 treatment (0.5 µg/ml) of SK-Mel-147 melanoma cells or HLEC (left and right graphs, respectively). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control.Quantification of the impact of BO-110 as single agent or in the presence of the indicated blocking antibodies for type I interferon (IFNB1 or IFNAR1). Upper graphs show the effect of these agents on MDK mRNA levels in SK-Mel-147 melanoma cells. Similar treatments were performed on HLEC for the analysis of VEGFR3 mRNA (middle graphs) and tube formation capacity (lower graphs). Data correspond to the mean ± SD of 3 biological replicates in triplicate.Growth of B16 melanoma xenografts in siblings of Ifnar1+/+, Ifnar1+/- or Ifnar1-/- mice. Treatment started 10 days after tumor cell implantation. BO-110 was administered at 0.8 mg/kg, every third day for 2 weeks. N= 6 mice per condition. Graphs show the mean tumor size ± SD at each time point. Statistical significance was determined by Two-Way ANOVA.


Figure 6Impact of BO - 110 on tumor - bearing mice implanted with syngeneic B16R2L ( 5 x 105 cells ) . When tumors have an average size on 100 mm3 , mice were randomized into two groups and treated with BO - 110 ( BO ) or vehicle ( V ) , every second day for 2 weeks . The arrow indicates the start of the treatment . Tumor size was measured with a caliper at the indicated time points . , N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA . Vegfr3Luc emission in the inguinal and brachial lymph nodes of mice in experiment ( A ) . N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA . G . ELISA analysis of Mdk blood levels in mice in experiment ( A ) . H . ELISA analysis of mouse Ifn - β blood levels in mice in experiment ( A ) . , N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA .

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Figure 6Impact of BO-110 on tumor-bearing mice implanted with syngeneic B16R2L (5 x 105 cells). When tumors have an average size on 100 mm3, mice were randomized into two groups and treated with BO-110 (BO) or vehicle (V), every second day for 2 weeks. The arrow indicates the start of the treatment. Tumor size was measured with a caliper at the indicated time points. , N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.Vegfr3Luc emission in the inguinal and brachial lymph nodes of mice in experiment (A). N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.G. ELISA analysis of Mdk blood levels in mice in experiment (A). H. ELISA analysis of mouse Ifn-β blood levels in mice in experiment (A). , N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.


Figure 7Efficacy of BO - 110 as an adjuvant ( preventing relapse after surgical removal of the primary lesion ) . Shown are representative examples of Vegfr3Luc mice implanted with mCherry - SK - Mel - 147 and imaged for luciferase emission ( prior to and after tumor removal . Animals were left to recover from surgery ( 4 days ) and then treated for 2 weeks ( 4 dosis ) with 0 . 8 mg / Kg BO - 110 or vehicle control ( n = 8 for control and n = 10 for treatment arm ) . Scale , p / s / cm2 / sr x106 . mCherry emission from tumor cells of the animals in ( A ) . Scale , p / s / cm2 / sr x109 . Kaplan - Meier survival curves of animals treated as in ( A ) . 8 / 8 animals treated with vehicle ( V ) control had to be sacrificed for humane reasons 110 days after surgery . 9 / 10 animals in the BO - 110 arm ( BO ) remained tumor - free 8 months after stopping treatment . The grey box marks the period of treatment with BO - 110 . Statistical significance was determined by logrank test .

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Figure 7Efficacy of BO-110 as an adjuvant (preventing relapse after surgical removal of the primary lesion). Shown are representative examples of Vegfr3Luc mice implanted with mCherry-SK-Mel-147 and imaged for luciferase emission (prior to and after tumor removal. Animals were left to recover from surgery (4 days) and then treated for 2 weeks (4 dosis) with 0.8 mg/Kg BO-110 or vehicle control (n= 8 for control and n=10 for treatment arm). Scale, p/s/cm2/sr x106.   mCherry emission from tumor cells of the animals in (A). Scale, p/s/cm2/sr x109.   Kaplan-Meier survival curves of animals treated as in (A). 8/8 animals treated with vehicle (V) control had to be sacrificed for humane reasons 110 days after surgery. 9/10 animals in the BO-110 arm (BO) remained tumor-free 8 months after stopping treatment. The grey box marks the period of treatment with BO-110. Statistical significance was determined by logrank test.


Figure 1 ( B ) Fluorescence microscopy images of typical mouse small intestinal organoids under different culture conditions . Lgr5 + intestinal stem cells are labeled with GFP , which are ubiquitously present , restricted to the bottom of the crypts and absent in CV , ENR and EN , respectively . * is auto - fluorescence

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Figure 1 (B) Fluorescence microscopy images of typical mouse small intestinal organoids under different culture conditions. Lgr5+ intestinal stem cells are labeled with GFP, which are ubiquitously present, restricted to the bottom of the crypts and absent in CV, ENR and EN, respectively. * is auto-fluorescence


Figure 2 ( A ) Two row - matched heatmaps showing the relative change in mRNA expression ( left ) and protein expression ( right ) of significantly changing proteins . Known markers of intestinal homeostasis are highlighted . Relative expression is shown as log2 fold change over the row mean

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Figure 2 (A) Two row-matched heatmaps showing the relative change in mRNA expression (left) and protein expression (right) of significantly changing proteins. Known markers of intestinal homeostasis are highlighted. Relative expression is shown as log2 fold change over the row mean


Figure 4 ( B ) Hematoxylin and ( Periodic acid - Shiff ) PAS staining of WT ( left ) and Hnf4g KO ( right ) intestine . Nuclei are visualized using Hematoxylin and goblet cells stain positive for PAS ( C ) Alcian Blue and Nuclear Fast Red staining of WT ( left ) and Hnf4g KO ( right ) small intestinal organoids . Cells are visualized using Nuclear Fast Red . Intra - and extracellular mucus that is produced in goblet cells stain positive for Alcian Blue ( D ) Strand specific RNA - seq data , Hnf4g ChIP - seq in EN , and promoter specific histone modification H3K4me3 over the Hnf4a locus is shown . Data from different organoid cultures are color coded . RNA - seq reads mapping to the positive strand ( sense strand ) are plotted in the top window of every sample , while RNA - seq reads that map to the negative strand are mirrored and shown in the bottom window of every sample . The two isoforms of Hnf4a that are expressed are shown in the bottom , together with the antisense transcript Hnf4aos . The " phyloP30way " track from the UCSC genome browser is plotted at the bottom to show sequence conservation ( E ) Quantification of the amount of goblet cells per villus for WT and Hnf4g KO intestine . Dots represent the amount of goblet cells counted per villus . ( n = 7 to 25 villi from 11 WT mice and 3 to 17 villi from 14 Hnf4g KO mice ) . P value is from two - tailed Mann - Whitney U test

O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 (B) Hematoxylin and (Periodic acid-Shiff) PAS staining of WT (left) and Hnf4g KO (right) intestine. Nuclei are visualized using Hematoxylin and goblet cells stain positive for PAS  (C) Alcian Blue and Nuclear Fast Red staining of WT (left) and Hnf4g KO (right) small intestinal organoids. Cells are visualized using Nuclear Fast Red. Intra- and extracellular mucus that is produced in goblet cells stain positive for Alcian Blue  (D) Strand specific RNA-seq data, Hnf4g ChIP-seq in EN, and promoter specific histone modification H3K4me3 over the Hnf4a locus is shown. Data from different organoid cultures are color coded. RNA-seq reads mapping to the positive strand (sense strand) are plotted in the top window of every sample, while RNA-seq reads that map to the negative strand are mirrored and shown in the bottom window of every sample. The two isoforms of Hnf4a that are expressed are shown in the bottom, together with the antisense transcript Hnf4aos. The "phyloP30way" track from the UCSC genome browser is plotted at the bottom to show sequence conservation  (E) Quantification of the amount of goblet cells per villus for WT and Hnf4g KO intestine. Dots represent the amount of goblet cells counted per villus. (n = 7 to 25 villi from 11 WT mice and 3 to 17 villi from 14 Hnf4g KO mice). P value is from two-tailed Mann-Whitney U test


Figure 1 A . HEK293 cells were treated for 8 and 24 h with 100 μM H2O2 . GSNOR was evaluated by Western blot . Densitometry of each lane is normalized to GADPH , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n ≥ 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 calculated with regard to H2O2 - untreated cells . B . HEK293 cells were treated for 8 h with 100 or 200 μM H2O2 . Nrf2 ( left panel ) and GSNOR ( right panel ) were evaluated by Western blot performed in nuclear and cytosol - enriched fractions . GAPDH and Lamin A / C were used as loading and purity controls of cytosol and nuclei , respectively . Densitometry of GSNOR immunoreactive bands is normalized to GADPH and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 . C . HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of the Nrf2 inhibitor trigonelline ( Trig ) . Heme oxygenase 1 ( HMOX - 1 ) , glutamate : cysteine ligase ( GCL ) and GSNOR expressions were evaluated RT - qPCR analyses Results shown are the means ± SEM of n = 3 experiments run in triplicate . * p < 0 . 05 ; n . s . , not significant . D . RT - qPCR analyses of GSNOR mRNA after 2 - to - 24 h incubation with 100 μM H2O2 . Results shown are the means ± SEM of n = 3 experiments run in triplicate , analyzed using ANOVA with Dunnett multiple comparisons test . n . s . , not significant . E . Western blot analysis of GSNOR in HEK293 cells treated for 8 h with 100 μM H2O2 in the presence or absence of trigonelline ( Trig ) . Densitometry of GSNOR immunoreactive bands is normalized to GADPH , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 . HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of cycloheximide ( CHX , F ) , GSNOR was evaluated by Western blot . Vertical dotted lines represent a virtual division of the nitrocellulose filter , as immunoreactive bands reported in figure - although part of the same experiment / gel - were not contiguous . Vehicle : PBS . Densitometry of GSNOR immunoreactive bands is normalized to Vinculin , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 4 independent experiments . * , p < 0 . 05 ; * * * , p < 0 . 001 ; n . s . , not significant . G . HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of the proteasome inhibitor MG132 ( G ) . GSNOR was evaluated by Western blot . Vertical dotted lines represent a virtual division of the nitrocellulose filter , as immunoreactive bands reported in figure - although part of the same experiment / gel - were not contiguous . Vehicle : PBS . Densitometry of GSNOR immunoreactive bands is normalized to Vinculin , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 4 independent experiments . * , p < 0 . 05 ; * * * , p < 0 . 001 ; n . s . , not significant . H . HEK293 cells were treated for 8 h ( blue ) or 24 h ( red ) with 100 μM H2O2 . Polysome profile showing monosomes and polysomes was obtained from control ( Vehicle ) and H2O2 treated lysates ( for either 8 or 24 h ) by separation on 5 - 50 % sucrose linear density gradient and collection using a gradient fractionation system . I . RT - qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2 - treated samples ( for either 8 or 24 h ) . H3A mRNA expression level was used as housekeeping gene control . Results represent the means ± SD of n ≥ 6 independent experiments shown as % normalized by H3A . * * * , p < 0 . 001 Vehicle : PBS . J . HEK293 cells treated for 8 h or 24 h with 100 μM H2O2 in the presence or absence of puromycin ( tyrosyl - tRNA mimic used to label nascent proteins ) , or cycloheximide ( CHX , used to block protein translation ) . Nascent polypeptides were assessed by Western blot performed with an anti - puromycin ( anti - PMC ) antibody . Vehicle : PBS . β - actin was used as loading control .

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

Figure 1 A. HEK293 cells were treated for 8 and 24 h with 100 μM H2O2. GSNOR was evaluated by Western blot. Densitometry of each lane is normalized to GADPH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n ≥ 3 independent experiments. *, p<0.05; **, p<0.01 calculated with regard to H2O2-untreated cells.  B. HEK293 cells were treated for 8 h with 100 or 200 μM H2O2. Nrf2 (left panel) and GSNOR (right panel) were evaluated by Western blot performed in nuclear and cytosol-enriched fractions. GAPDH and Lamin A/C were used as loading and purity controls of cytosol and nuclei, respectively. Densitometry of GSNOR immunoreactive bands is normalized to GADPH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.  C. HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of the Nrf2 inhibitor trigonelline (Trig). Heme oxygenase 1 (HMOX-1), glutamate:cysteine ligase (GCL) and GSNOR expressions were evaluated RT-qPCR analyses Results shown are the means ± SEM of n = 3 experiments run in triplicate. *p<0.05; n.s., not significant.  D. RT-qPCR analyses of GSNOR mRNA after 2-to-24 h incubation with 100 μM H2O2. Results shown are the means ± SEM of n = 3 experiments run in triplicate, analyzed using ANOVA with Dunnett multiple comparisons test. n.s., not significant.  E. Western blot analysis of GSNOR in HEK293 cells treated for 8 h with 100 μM H2O2 in the presence or absence of trigonelline (Trig). Densitometry of GSNOR immunoreactive bands is normalized to GADPH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01.  HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of cycloheximide (CHX, F), GSNOR was evaluated by Western blot. Vertical dotted lines represent a virtual division of the nitrocellulose filter, as immunoreactive bands reported in figure - although part of the same experiment/gel - were not contiguous. Vehicle: PBS. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 4 independent experiments. *, p<0.05; ***, p<0.001; n.s., not significant.  G. HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of the proteasome inhibitor MG132 (G). GSNOR was evaluated by Western blot. Vertical dotted lines represent a virtual division of the nitrocellulose filter, as immunoreactive bands reported in figure - although part of the same experiment/gel - were not contiguous. Vehicle: PBS. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 4 independent experiments. *, p<0.05; ***, p<0.001; n.s., not significant.  H. HEK293 cells were treated for 8 h (blue) or 24 h (red) with 100 μM H2O2. Polysome profile showing monosomes and polysomes was obtained from control (Vehicle) and H2O2 treated lysates (for either 8 or 24 h) by separation on 5-50% sucrose linear density gradient and collection using a gradient fractionation system.  I. RT-qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2-treated samples (for either 8 or 24 h). H3A mRNA expression level was used as housekeeping gene control. Results represent the means ± SD of n ≥ 6 independent experiments shown as % normalized by H3A. ***, p<0.001 Vehicle: PBS.  J. HEK293 cells treated for 8 h or 24 h with 100 μM H2O2 in the presence or absence of puromycin (tyrosyl-tRNA mimic used to label nascent proteins), or cycloheximide (CHX, used to block protein translation). Nascent polypeptides were assessed by Western blot performed with an anti-puromycin (anti-PMC) antibody. Vehicle: PBS. β-actin was used as loading control.


Figure 2 A . HEK293 cells treated for 24 h with 100 μM H2O2 . Basal and phospho - active forms of ATM and CHK2 were assessed by Western blot . Phospho : basal level ratios of ATM and CHK2 immunoreactive bands are normalized to Vinculin , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n ≥ 4 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 . B . HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 ( KU ) . Basal and phospho - active forms of ATM , CHK2 , and GSNOR were assessed by Western blot . Vinculin and GAPDH were used as loading controls . Phospho : basal level ratios of ATM and CHK2 ( normalized to Vinculin ) , along with densitometry of GSNOR immunoreactive bands ( normalized to GAPDH ) are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 . C . HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the CHK2 inhibitor AZD7762 ( AZD ) . Basal and phospho - active forms of CHK2 , together with GSNOR were assesses by Western blot . Phospho : basal level ratios of CHK2 and densitometry of GSNOR immunoreactive bands are normalized to GAPDH , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant . D . HEK293 cells were transfected with a pool of siRNA against ATM ( siATM ) or with control siRNA ( siScr ) for 18 h and treated for additional 24 h with 100 μM H2O2 . Basal and phospho - active forms of ATM , together with GSNOR were assessed by Western blot . Vinculin and GAPDH were used as loading controls . Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 . E . HEK293 cells were transfected with a pool of siRNA against CHK2 ( siCHK2 ) or with control siRNA ( siScr ) for 18 h and treated for additional 24 h with 100 μM H2O2 . Basal and phospho - active forms of CHK2 , together with GSNOR were assessed by Western blot . GAPDH was used as loading control . Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 . F . HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 ( KU ) . Lysates were subjected to biotin - switch assay , and S - nitrosylated proteins ( PSNOs ) revealed by incubation with horseradish peroxidase ( HRP ) - conjugated streptavidin . Results obtained in the absence of ascorbate ( - ASC ) are shown as negative controls . Densitometry of each lane intensity is normalized to LDH , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 . G . RT - qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2 treated samples in the absence or in the presence of the ATM inhibitor KU55933 ( KU ) . H3A mRNA expression level was used as housekeeping gene control . Results are the means ± SEM of n = 3 independent experiments run in duplicate and shown as % , normalized by H3A . * , p < 0 . 05 . H . HEK293 cells were treated for 8 or 24 h with 100 μM H2O2 or , alternatively , for 2 h with 2 mM hydroxyurea ( HU ) , selected as positive control of CHK1 phosphorylation at Ser317 . Phospho - CHK1Ser317 was assessed by Western blot ; densitometry normalized to Vinculin , selected as loading control , and expressed as arbitrary units ( with HU arbitrarily set to 1 ) . Values shown represent the means ± SD of n = 3 independent experiments . n . s . , not significant . I . HEK293 cells were transfected with a pool of siRNA against CHK1 ( siCHK1 ) or with control siRNA ( siScr ) for 18 h and treated for additional 24 h with 100 μM H2O2 . CHK1 and GSNOR were assessed by Western blot analysis of . Densitometry of GSNOR immunoreactive bands is normalized to Vinculin , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 .

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

Figure 2 A. HEK293 cells treated for 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2 were assessed by Western blot. Phospho:basal level ratios of ATM and CHK2 immunoreactive bands are normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n ≥ 4 independent experiments. *, p<0.05; **, p<0.01.  B. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 (KU). Basal and phospho-active forms of ATM, CHK2, and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Phospho:basal level ratios of ATM and CHK2 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.  C. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the CHK2 inhibitor AZD7762 (AZD). Basal and phospho-active forms of CHK2, together with GSNOR were assesses by Western blot. Phospho:basal level ratios of CHK2 and densitometry of GSNOR immunoreactive bands are normalized to GAPDH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.  D. HEK293 cells were transfected with a pool of siRNA against ATM (siATM) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM, together with GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01.  E. HEK293 cells were transfected with a pool of siRNA against CHK2 (siCHK2) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of CHK2, together with GSNOR were assessed by Western blot. GAPDH was used as loading control. Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.  F. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 (KU). Lysates were subjected to biotin-switch assay, and S-nitrosylated proteins (PSNOs) revealed by incubation with horseradish peroxidase (HRP)-conjugated streptavidin. Results obtained in the absence of ascorbate (- ASC) are shown as negative controls. Densitometry of each lane intensity is normalized to LDH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01.  G. RT-qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2 treated samples in the absence or in the presence of the ATM inhibitor KU55933 (KU). H3A mRNA expression level was used as housekeeping gene control. Results are the means ± SEM of n = 3 independent experiments run in duplicate and shown as %, normalized by H3A. *, p<0.05.  H. HEK293 cells were treated for 8 or 24 h with 100 μM H2O2 or, alternatively, for 2 h with 2 mM hydroxyurea (HU), selected as positive control of CHK1 phosphorylation at Ser317. Phospho-CHK1Ser317 was assessed by Western blot; densitometry normalized to Vinculin, selected as loading control, and expressed as arbitrary units (with HU arbitrarily set to 1). Values shown represent the means ± SD of n = 3 independent experiments. n.s., not significant.  I. HEK293 cells were transfected with a pool of siRNA against CHK1 (siCHK1) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. CHK1 and GSNOR were assessed by Western blot analysis of. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.


Figure 3 A . HEK293 cells treated for 4 h with 100 μM H2O2 . Phospho - histone H2A . X ( γH2A . X ) was assessed by immunofluorescence analysis . Nuclei ( blue ) were stained with Hoechst 33342 . scale bar : 10μM . 4X digital magnification is shown at the bottom left of each image . B . Additionally , γH2A . X was also assessed by Western blot at the indicated time points . GAPDH was used as loading control ( top panel ) . Densitometry of each lane intensity is normalized to GAPDH , selected as loading control , and expressed as % of γH2A . X in H2O2 - treated versus untreated cells . Values represent the means ± SD of two independent experiments ( bottom panel ) . C . HEK293 cells were treated for 8 or 24 h with 100 μM H2O2 or , alternatively , for 2 h with 2 mM hydroxyurea ( HU ) , selected as positive control of ATR / CHK1 axis activation . Basal and phospho - active forms of ATR and CHK1 ( at Ser345 ) were assessed by Western blot . GAPDH was used as loading control . D . HEK293 cells were treated for 8 h with 20 μM neocarzinostatin ( NCS ) . ( left ) Western blot analysis of basal and phospho - active forms of ATM , CHK2 , ATR , CHK1 , p53 , and GSNOR in HEK293 cells treated for 8 h with 20 μM neocarzinostatin ( NCS ) . Vinculin and GAPDH were used as loading controls . ( right ) Phospho : basal level ratios of ATM , CHK2 , ATR , CHK1 , along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant . E . HEK293 cells were treated for 2 , 4 and 8 h with 20 μM neocarzinostatin ( NCS ) . After treatment , cells were incubated with 2 ' , 7 ' - H2DCF - DA to cytofluorometrically assess the intracellular production of H2O2 . Values are shown as units of DCF fluorescence relative to untreated cells . Values are shown as units of fluorescence relative to NCS - untreated cells ( arbitrarily set as 1 ) and represent the means ± SD of n = 3 independent experiments . n . s . , not significant . F . HEK293 cells were transfected with plasmids coding for the wild - type ( WT ) , C2991L redox mutant ( CL ) of ATM , or with an empty vector ( Empty ) ( used as negative control ) for 40 h and treated for additional 8 h with 100 μM H2O2 . Basal and phospho - active forms of ATM , and GSNOR were assessed by Western blot analysis . Vinculin and GAPDH were used as loading controls . Two different exposures ( low and high ) were selected to highlight differences in P - ATM levels in different experimental settings .

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

Figure 3 A. HEK293 cells treated for 4 h with 100 μM H2O2. Phospho-histone H2A.X (γH2A.X) was assessed by immunofluorescence analysis. Nuclei (blue) were stained with Hoechst 33342. scale bar: 10μM. 4X digital magnification is shown at the bottom left of each image.  B. Additionally, γH2A.X was also assessed by Western blot at the indicated time points. GAPDH was used as loading control (top panel). Densitometry of each lane intensity is normalized to GAPDH, selected as loading control, and expressed as % of γH2A.X in H2O2-treated versus untreated cells. Values represent the means ± SD of two independent experiments (bottom panel).  C. HEK293 cells were treated for 8 or 24 h with 100 μM H2O2 or, alternatively, for 2 h with 2 mM hydroxyurea (HU), selected as positive control of ATR/CHK1 axis activation. Basal and phospho-active forms of ATR and CHK1 (at Ser345) were assessed by Western blot. GAPDH was used as loading control.  D. HEK293 cells were treated for 8 h with 20 μM neocarzinostatin (NCS). (left) Western blot analysis of basal and phospho-active forms of ATM, CHK2, ATR, CHK1, p53, and GSNOR in HEK293 cells treated for 8 h with 20 μM neocarzinostatin (NCS). Vinculin and GAPDH were used as loading controls. (right) Phospho:basal level ratios of ATM, CHK2, ATR, CHK1, along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant. E. HEK293 cells were treated for 2, 4 and 8 h with 20 μM neocarzinostatin (NCS). After treatment, cells were incubated with 2',7'-H2DCF-DA to cytofluorometrically assess the intracellular production of H2O2. Values are shown as units of DCF fluorescence relative to untreated cells. Values are shown as units of fluorescence relative to NCS-untreated cells (arbitrarily set as 1) and represent the means ± SD of n = 3 independent experiments. n.s., not significant.  F. HEK293 cells were transfected with plasmids coding for the wild-type (WT), C2991L redox mutant (CL) of ATM, or with an empty vector (Empty) (used as negative control) for 40 h and treated for additional 8 h with 100 μM H2O2. Basal and phospho-active forms of ATM, and GSNOR were assessed by Western blot analysis. Vinculin and GAPDH were used as loading controls. Two different exposures (low and high) were selected to highlight differences in P-ATM levels in different experimental settings.


Figure 4 A . HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of 20 μM pifithrin - α ( Pft ) . Basal and phospho - active forms of ATM , CHK2 , p53 , and GSNOR were assessed by Western blot . Phospho : basal level ratios of ATM , CHK2 and p53 ( normalized to Vinculin ) , along with densitometry of GSNOR immunoreactive bands ( normalized to GAPDH ) are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant . B . HEK293 cells were transfected with a pool of siRNA against p53 ( sip53 ) or with control siRNA ( siScr ) for 24 h and treated for additional 24 h with 100 μM H2O2 . Basal and phospho - active forms of ATM and CHK2 , as well as p53 and GSNOR were assessed by Western blot . Phospho : basal level ratios of ATM and CHK2 ( normalized to Vinculin ) , along with densitometry of GSNOR immunoreactive bands ( normalized to GAPDH ) are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant . C . HCT116 cells expressing the wild type form of p53 ( p53wt ) , or an empty vector ( Empty , selected as negative control ) , were treated for 24 h with 100 μM H2O2 . Basal and phospho - active forms of ATM and CHK2 , p53 and GSNOR were assessed by Western blot . Vinculin and GAPDH were used as loading controls . Phospho : basal level ratios of ATM and CHK2 , along with densitometry of p53 and GSNOR immunoreactive bands are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

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

Figure 4 A. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of 20 μM pifithrin-α (Pft). Basal and phospho-active forms of ATM, CHK2, p53, and GSNOR were assessed by Western blot. Phospho:basal level ratios of ATM, CHK2 and p53 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.  B. HEK293 cells were transfected with a pool of siRNA against p53 (sip53) or with control siRNA (siScr) for 24 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, as well as p53 and GSNOR were assessed by Western blot. Phospho:basal level ratios of ATM and CHK2 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.  C. HCT116 cells expressing the wild type form of p53 (p53wt), or an empty vector (Empty, selected as negative control), were treated for 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, p53 and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Phospho:basal level ratios of ATM and CHK2, along with densitometry of p53 and GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.


Figure 5 A , B . HEK293 cells treated for 2 , 4 and 8 h with 100 μM H2O2 . After treatment , cells were incubated with 5 μM 2 ' , 7 ' - H2DCF - DA ( A ) or MitoSox ( B ) to evaluate the production of H2O2 or mitochondrial superoxide , respectively . Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells ( arbitrarily set as 1 ) and represent the means ± SD of n ≥ 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . C . HEK293 were transfected for 48 h with siRNA against ATM ( siATM ) , GSNOR ( siGSNOR ) , or control siRNA ( scramble , siScr ) . Afterwards , they were treated for 8 h with 100 μM H2O2 and mitophagy was assessed by Western blot of different mitochondrial complex subunits [ i . e . , NDUFB8 ( complex I ) , SDHB ( complex II ) , MTCO2 ( complex IV ) ] . Basal and phospho ATM , as well as GSNOR were used to check the efficiency of siRNA - mediated knock down . Vinculin was used as loading control . D . Densitometry of mitochondrial protein immunoreactive bands of panel C ( normalized to Vinculin ) is indicated as H2O2 - treated versus untreated cells ( CTR ) and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . , not significant . E . In the same experimental settings , mitophagy was also assessed by RT - qPCR relative quantitation of D - loop ( selected as measure of mtDNA ) normalized to genomic actin ( gActin ) . Results shown are the means ± SD of n = 8 experiments * * * p < 0 . 001 ; n . s . , not significant . F . U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 4 h with 100 μM H2O2 . Mitophagy was assessed by RT - qPCR relative quantitation of D - loop normalized to genomic actin ( gActin ) . Results shown are the means ± SD of n = 6 experiments . * p < 0 . 05 ; * * * p < 0 . 001 . In the same experimental settings , mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine ( CLQ ) to enhance differences in mitophagy . Percentage of mitochondria merging with LC3 - positive puncta calculated by Fiji analysis software using the open - source plugin ComDet v . 0 . 3 . 7 . Values are expressed as % of mitochondria ( TOM20 + particles ) co - localizing with LC3 / cell and graphed as boxes ( 25th - 75th interquartile range ) and whiskers ( minimum to maximum showing all points ) , with central bands representing the median of n ≥ 7 different cells analyzed . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . H . In the same experimental settings , mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine ( CLQ ) to enhance differences in mitophagy . Anti - TOM20 ( red ) was used to visualize mitochondria ; anti - LC3 ( green ) was used to identify autophagosomes .

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

Figure 5 A, B. HEK293 cells treated for 2, 4 and 8 h with 100 μM H2O2. After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or MitoSox (B) to evaluate the production of H2O2 or mitochondrial superoxide, respectively. Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells (arbitrarily set as 1) and represent the means ± SD of n ≥ 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.  C. HEK293 were transfected for 48 h with siRNA against ATM (siATM), GSNOR (siGSNOR), or control siRNA (scramble, siScr). Afterwards, they were treated for 8 h with 100 μM H2O2 and mitophagy was assessed by Western blot of different mitochondrial complex subunits [i.e., NDUFB8 (complex I), SDHB (complex II), MTCO2 (complex IV)]. Basal and phospho ATM, as well as GSNOR were used to check the efficiency of siRNA-mediated knock down. Vinculin was used as loading control.  D. Densitometry of mitochondrial protein immunoreactive bands of panel C (normalized to Vinculin) is indicated as H2O2-treated versus untreated cells (CTR) and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant.  E. In the same experimental settings, mitophagy was also assessed by RT-qPCR relative quantitation of D-loop (selected as measure of mtDNA) normalized to genomic actin (gActin). Results shown are the means ± SD of n = 8 experiments ***p<0.001; n.s., not significant.  F. U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 4 h with 100 μM H2O2. Mitophagy was assessed by RT-qPCR relative quantitation of D-loop normalized to genomic actin (gActin). Results shown are the means ± SD of n = 6 experiments. *p<0.05; ***p<0.001.  In the same experimental settings, mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Percentage of mitochondria merging with LC3-positive puncta calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. Values are expressed as % of mitochondria (TOM20+ particles) co-localizing with LC3/cell and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n ≥ 7 different cells analyzed. *p<0.05; **p<0.01; ***p<0.001.  H. In the same experimental settings, mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Anti-TOM20 (red) was used to visualize mitochondria; anti-LC3 (green) was used to identify autophagosomes.


Figure 6 A , B . ATMWT U2OS cells were treated with 10 μM CCCP for 8 h . After treatment , cells were incubated with 5 μM 2 ' , 7 ' - H2DCF - DA ( A ) or MitoSox ( B ) to evaluate the production of H2O2 or mitochondrial superoxide , respectively . Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells ( arbitrarily set as 1 ) and represent the means ± SD of n = 3 independent experiments . * * * p < 0 . 001 . C . In the same experimental settings , basal and phospho - active forms of ATM , and GSNOR were assessed by Western blot . Vinculin and GAPDH were used as loading controls . U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 2 h with 10 μM CCCP . Mitophagy was evaluated by : ( D ) RT - qPCR relative quantitation of D - loop normalized to genomic actin ( gActin ) . Results shown are the means ± SD of n = 4 experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 2 h with 10 μM CCCP . ( E fluorescence microscopy analyses upon incubation with chloroquine ( CLQ ) to enhance differences in mitophagy . Anti - TOM20 ( red ) was used to visualize mitochondria ; anti - LC3 ( green ) was used to identify autophagosomes . U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 2 h with 10 μM CCCP . F ) fluorescence microscopy analyses upon incubation with chloroquine ( CLQ ) to enhance differences in mitophagy . Percentage of mitochondria merging with LC3 - positive puncta calculated by Fiji analysis software using the open - source plugin ComDet v . 0 . 3 . 7 . Values are expressed as % of mitochondria ( TOM20 + particles ) co - localizing with LC3 / cell and graphed as boxes ( 25th - 75th interquartile range ) and whiskers ( minimum to maximum showing all points ) , with central bands representing the median of n ≥ 9 different cells . * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . , not significant . U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 2 h with 10 μM CCCP . ( G Western blot of different subunits of mitochondrial proteins , i . e . , NDUFB8 ( complex I ) , SDHA and SDHB ( complex II ) , MTCO2 ( complex IV ) and voltage - dependent anion channel ( VDAC ) . Tubulin , LDH and Vinculin were used as loading controls . U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , cells were further transfected with a GSNOR - coding vector and then treated for 2 h with 10 μM CCCP . H ) Densitometry of mitochondrial protein immunoreactive bands and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . , not significant .

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

Figure 6 A, B. ATMWT U2OS cells were treated with 10 μM CCCP for 8 h. After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or MitoSox (B) to evaluate the production of H2O2 or mitochondrial superoxide, respectively. Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells (arbitrarily set as 1) and represent the means ± SD of n = 3 independent experiments. ***p< 0.001.  C. In the same experimental settings, basal and phospho-active forms of ATM, and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls.  U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. Mitophagy was evaluated by: (D) RT-qPCR relative quantitation of D-loop normalized to genomic actin (gActin). Results shown are the means ± SD of n = 4 experiments. *p<0.05; **p<0.01; ***p<0.001.  U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. (E fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Anti-TOM20 (red) was used to visualize mitochondria; anti-LC3 (green) was used to identify autophagosomes.  U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. F) fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Percentage of mitochondria merging with LC3-positive puncta calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. Values are expressed as % of mitochondria (TOM20+ particles) co-localizing with LC3/cell and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n ≥ 9 different cells. **p<0.01; ***p<0.001; n.s., not significant.  U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. (G Western blot of different subunits of mitochondrial proteins, i.e., NDUFB8 (complex I), SDHA and SDHB (complex II), MTCO2 (complex IV) and voltage-dependent anion channel (VDAC). Tubulin, LDH and Vinculin were used as loading controls.  U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. H) Densitometry of mitochondrial protein immunoreactive bands and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant.


Figure 7 B . HEK293 cells overexpressing the wild type form of GSNOR ( GSNORwt ) or an empty vector ( Empty ) were subjected to combined treatment ( 200 μM H2O2 + 400 μM DPTA ) in the presence or absence of the ATM inhibitor KU55933 ( KU ) . Analysis of dead cells were performed with Trypan blue exclusion assay . Western blot analysis of GSNOR is shown as inset in the graph to substantiate transfection efficiency . Data , shown as fold change of dead cells relative to untreated cells ( arbitrarily set to 1 ) , represent the mean count ± SEM of n = 4 experiments done in duplicate * p < 0 . 05 ; with respect to Empty cells . C . Representative optic microscopy image of GSNORwt and Empty cells upon 24 h treatment with H2O2 + DPTA in the presence KU . D . Western blot analysis of Caspase3 and PARP1 in GSNORwt and Empty cells treated as in panel C . Tubulin was used as loading control . E . HEK293 cells overexpressing the wild type form of GSNOR ( GSNORwt ) or an empty vector ( Empty ) were transfected with siRNA against Pakin ( siParkin ) or control siRNA ( scramble , siScr ) . Afterwards , they were subjected to combined treatment ( 200 μM H2O2 + 400 μM DPTA ) and viability assessed by Alamar blue ( AB ) fluorescent assay . Data , shown as fold change of viable cells , refer to AB fluorescence ( relative to untreated cells , arbitrarily set to 1 ) and represent the means ± SD of n = 6 independent experiments . * * , p < 0 . 01 ; n . s . , not significant . F . Western blot of Gsnor was assessed in mouse adult fibroblasts ( MAFs ) , obtained from wild type ( WT ) and Gsnor - null ( KO ) mice treated with 200 or 400 μM H2O2 for 24 h . WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2 , or 400 μM DPTA , or a combination of both . Cell viability was evaluated by LIVE / DEAD assay . H . WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2 , or 400 μM DPTA , or a combination of both . Cell viability was evaluated by LIVE / DEAD assay . Scale bar = 50 µm . Data , shown as % of dead ( red ) cells , represent the mean count ± SD of n = 3 different fields of three independent experiments . * p < 0 . 05 ; n . s . , not significant with respect to WT MAFs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7 B. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) in the presence or absence of the ATM inhibitor KU55933 (KU). Analysis of dead cells were performed with Trypan blue exclusion assay. Western blot analysis of GSNOR is shown as inset in the graph to substantiate transfection efficiency. Data, shown as fold change of dead cells relative to untreated cells (arbitrarily set to 1), represent the mean count ± SEM of n = 4 experiments done in duplicate *p<0.05; with respect to Empty cells.  C. Representative optic microscopy image of GSNORwt and Empty cells upon 24 h treatment with H2O2 + DPTA in the presence KU. D. Western blot analysis of Caspase3 and PARP1 in GSNORwt and Empty cells treated as in panel C. Tubulin was used as loading control. E. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were transfected with siRNA against Pakin (siParkin) or control siRNA (scramble, siScr). Afterwards, they were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) and viability assessed by Alamar blue (AB) fluorescent assay. Data, shown as fold change of viable cells, refer to AB fluorescence (relative to untreated cells, arbitrarily set to 1) and represent the means ± SD of n = 6 independent experiments. **, p<0.01; n.s., not significant.  F. Western blot of Gsnor was assessed in mouse adult fibroblasts (MAFs), obtained from wild type (WT) and Gsnor-null (KO) mice treated with 200 or 400 μM H2O2 for 24 h.  WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2, or 400 μM DPTA, or a combination of both. Cell viability was evaluated by LIVE/DEAD assay.  H. WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2, or 400 μM DPTA, or a combination of both. Cell viability was evaluated by LIVE/DEAD assay. Scale bar = 50 µm. Data, shown as % of dead (red) cells, represent the mean count ± SD of n = 3 different fields of three independent experiments. *p<0.05; n.s., not significant with respect to WT MAFs.


Figure 8 A , B . Jurkat cells were treated for 10 and 30 min with PMA ( 200 ng / ml ) and ionomycin ( iono ) ( 300 ng / ml ) . After treatment , cells were incubated with 5 μM 2 ' , 7 ' - H2DCF - DA ( A ) or DAF - FM - DA ( B ) to evaluate the production of H2O2 or NO , respectively . Values are expressed as units of DCF or DAF - FM fluorescence relative to untreated cells ( arbitrarily set as 1 ) and graphed as boxes ( 25th - 75th interquartile range ) and whiskers ( minimum to maximum showing all points ) , with central bands representing the median of n = 4 ( A ) and n = 6 ( B ) independent experiments . * p < 0 . 01 ; * * p < 0 . 001 ; * * * p < 0 . 001 . C . ( left ) Jurkat cells were treated for 24 h with H2O2 ( 50 μM ) or PMA / ionomycin ( 200 + 300 ng / ml ) . Basal and phospho - active form of ATM , and GSNOR were assessed by Western blot . Vinculin was used as loading control . ( right ) Phospho : basal level ratios of ATM along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 . D . Jurkat cells were treated for 24 h with H2O2 ( 50 μM ) or PMA / ionomycin ( 200 + 300 ng / ml ) , in the presence or absence of ATM inhibitor ( KU55933 ) ( 5 μM ) or GSNOR inhibitor ( N6022 ) ( 25 μM ) . Cell death was assessed cytofluorimetrically upon staining with propidium iodide ( PI ) . Values are expressed as % of sub - G1 population of PI - stained cells and graphed as boxes ( 25th - 75th interquartile range ) and whiskers ( minimum to maximum showing all points ) , with central bands representing the median of n = 5 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 . E , F . CD4 + T cells were incubated for 30 min with anti - CD3 , anti - CD28 and anti - CD49d ( stimulation ) . After stimulation , cells were incubated with 5 μM 2 ' , 7 ' - H2DCF - DA ( E ) or DAF - FM - DA ( F ) to fluorometrically evaluate the production of H2O2 or NO , respectively . Values are shown as units of DCF or DAF - FM fluorescence relative to non - stimulated cells ( arbitrarily set as 1 ) . Values are shown as fold change and represent the median plus range with all the experimental points of n = 3 independent experiments . * * p < 0 . 01 . G . CD4 + T cells were stimulated for 96 h with anti - CD3 , anti - CD28 and anti - CD49d in the presence or absence of ATM inhibitor ( KU , upper panel ) or GSNOR inhibitor ( N6022 , bottom panel ) . Cell death was assessed cytofluorometrically upon staining with Annexin V ( AnV ) and PI . Values are expressed as fold change of AnV + / PI + cells relative to control ( CD4 + without inhibitor , vehicle , arbitrarily set to 1 ) and graphed as boxes ( 25th - 75th interquartile range ) and whiskers ( minimum to maximum showing all points ) , with central bands representing the median of n = 4 independent experiments . * p < 0 . 05 ; * * * p < 0 . 001 . H . In the same experimental settings , populations of stimulated ( proliferating ) CD4 + T cells were identified cytofluorometrically and included in rectangles . Values ( as % of total population ) are shown in red in each representative plot identify and summed up in a graph as the means ± SD of n = 3 independent experiments . * p < 0 . 05 ; * * * p < 0 . 001 .

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

Figure 8 A, B. Jurkat cells were treated for 10 and 30 min with PMA (200 ng/ml) and ionomycin (iono) (300 ng/ml). After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or DAF-FM-DA (B) to evaluate the production of H2O2 or NO, respectively. Values are expressed as units of DCF or DAF-FM fluorescence relative to untreated cells (arbitrarily set as 1) and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 4 (A) and n = 6 (B) independent experiments. *p<0.01; **p<0.001; ***p<0.001.  C. (left) Jurkat cells were treated for 24 h with H2O2 (50 μM) or PMA/ionomycin (200 + 300 ng/ml). Basal and phospho-active form of ATM, and GSNOR were assessed by Western blot. Vinculin was used as loading control. (right) Phospho:basal level ratios of ATM along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.  D. Jurkat cells were treated for 24 h with H2O2 (50 μM) or PMA/ionomycin (200 + 300 ng/ml), in the presence or absence of ATM inhibitor (KU55933) (5 μM) or GSNOR inhibitor (N6022) (25 μM). Cell death was assessed cytofluorimetrically upon staining with propidium iodide (PI). Values are expressed as % of sub-G1 population of PI-stained cells and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 5 independent experiments. *p<0.05; **p<0.01.  E, F. CD4+ T cells were incubated for 30 min with anti-CD3, anti-CD28 and anti-CD49d (stimulation). After stimulation, cells were incubated with 5 μM 2',7'-H2DCF-DA (E) or DAF-FM-DA (F) to fluorometrically evaluate the production of H2O2 or NO, respectively. Values are shown as units of DCF or DAF-FM fluorescence relative to non-stimulated cells (arbitrarily set as 1). Values are shown as fold change and represent the median plus range with all the experimental points of n = 3 independent experiments. **p < 0.01.  G. CD4+ T cells were stimulated for 96 h with anti-CD3, anti-CD28 and anti-CD49d in the presence or absence of ATM inhibitor (KU, upper panel) or GSNOR inhibitor (N6022, bottom panel). Cell death was assessed cytofluorometrically upon staining with Annexin V (AnV) and PI. Values are expressed as fold change of AnV+/PI+ cells relative to control (CD4+ without inhibitor, vehicle, arbitrarily set to 1) and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 4 independent experiments. *p<0.05; ***p<0.001.  H. In the same experimental settings, populations of stimulated (proliferating) CD4+ T cells were identified cytofluorometrically and included in rectangles. Values (as % of total population) are shown in red in each representative plot identify and summed up in a graph as the means ± SD of n = 3 independent experiments. *p<0.05; ***p<0.001.


Figure 1 ( C ) T2 - weighted MR images in coronal plane show abscess formation in the left lower pulmonary lobe ( upper panel ) and in spleen and left axilla ( lower panel ) . ( D ) Histological sections of patient biopsies stained with hematoxylin and eosin ( left panel ) or with an actin antibody ( right panel ) . Scale bars : skin : left 1000µm , right 100µm ; lung : left 500µm , right 250µm ; spleen : left 1000µm , right 75µm .

O O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C) T2-weighted MR images in coronal plane show abscess formation in the left lower pulmonary lobe (upper panel) and in spleen and left axilla (lower panel).(D) Histological sections of patient biopsies stained with hematoxylin and eosin (left panel) or with an actin antibody (right panel). Scale bars: skin: left 1000µm, right 100µm; lung: left 500µm, right 250µm; spleen: left 1000µm, right 75µm.


Figure 2 ( A ) Expression of indicated proteins was determined by western blot in patient - derived and control fibroblasts and B cells and A549 OTULIN WT and KO cells . One representative out of three independent experiments is shown . ( B ) FLAG - tagged tandem ubiquitin binding entity ( TUBE ) assay was performed to pull down linear ubiquitin linkages in patient - derived and control fibroblasts and B cells . One representative out of three independent experiments is shown .

O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Expression of indicated proteins was determined by western blot in patient-derived and control fibroblasts and B cells and A549 OTULIN WT and KO cells. One representative out of three independent experiments is shown.(B) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed to pull down linear ubiquitin linkages in patient-derived and control fibroblasts and B cells. One representative out of three independent experiments is shown.


Figure 4Biochemical characterization of recombinant OTULIN variants by means of differential scanning fluorimetry ( DSF ) A ) DSF measurements with OTULINWT , OTULINM86I or OTULINW167S . Melting temperatures ( Tm ) are calculated from five independent experiments with standard deviations . * , P = 0 . 011 ; * * * * , P = 6 . 64x10 - 18 , unpaired t - test . Biochemical characterization of recombinant OTULIN variants by means of surface plasmon resonance ( SPR ) measurements ( B ) SPR measurements and steady - state binding curves with calculated dissociation constants ( Kd ) after injection of a concentration series of OTULINC129A , OTULINC129A / M86I or OTULINC129A / W167S to CM5 - immobilized di - ubiquitin chains . ( C ) Linear ubiquitin linkages isolated from A549 OTULIN KO cells by M1 TUBE assay were incubated with increasing concentrations of recombinant OTULINWT , OTULINM86I and OTULINW167S or catalytically inactive OTULINC129A as control for 1 hour . Afterwards , samples were subjected to analysis by western blot for the indicated proteins . Images are representative of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Biochemical characterization of recombinant OTULIN variants by means of differential scanning fluorimetry (DSF) A) DSF measurements with OTULINWT, OTULINM86I or OTULINW167S. Melting temperatures (Tm) are calculated from five independent experiments with standard deviations. *, P = 0.011; ****, P = 6.64x10-18, unpaired t-test.Biochemical characterization of recombinant OTULIN variants by means of surface plasmon resonance (SPR) measurements (B) SPR measurements and steady-state binding curves with calculated dissociation constants (Kd) after injection of a concentration series of OTULINC129A, OTULINC129A/M86I or OTULINC129A/W167S to CM5-immobilized di-ubiquitin chains.(C) Linear ubiquitin linkages isolated from A549 OTULIN KO cells by M1 TUBE assay were incubated with increasing concentrations of recombinant OTULINWT, OTULINM86I and OTULINW167S or catalytically inactive OTULINC129A as control for 1 hour. Afterwards, samples were subjected to analysis by western blot for the indicated proteins. Images are representative of three independent experiments.


Figure 5 ( A - C ) A549 OTULIN KO cells were transfected with empty vector or different OTULIN constructs as indicated and analyzed by western blot . ( D ) FLAG - tagged tandem ubiquitin binding entity ( TUBE ) assay was performed in B cells from control , patient , mother and father to pull down linear ubiquitin linkages . One representative ( A - D ) of three independent experiments is shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A - C) A549 OTULIN KO cells were transfected with empty vector or different OTULIN constructs as indicated and analyzed by western blot. (D) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed in B cells from control, patient, mother and father to pull down linear ubiquitin linkages. One representative (A - D) of three independent experiments is shown.


Figure 6Patient - derived and control fibroblasts were used . ( A ) FLAG - tagged tandem ubiquitin binding entity ( TUBE ) assay was performed to pull down linear ubiquitin linkages upon stimulation with 500ng / ml TNF . ( B ) The TNFR1 - SC was isolated using 500ng / ml TAP - TNF . ( C ) Cells were stimulated with 100ng / ml TNF for the indicated times and analyzed by western blot . One representative ( A - C ) of three independent experiments is shown . ( E ) B cells were incubated with 50µg / ml cycloheximide ( CHX ) for the indicated times and subjected to analysis by western blot . One representative ( left panel ) out of 4 individual experiments analyzed by densitometry ( right panel ) is shown . Data are presented as mean ± SD . * , P = 0 . 02 , unpaired t - test . ( G ) Fibroblasts were incubated with 50µg / ml cycloheximide ( CHX ) or DMSO and stimulated with TNF as indicated . Viability was measured after 24 hours . Data are presented as mean ± SD of 5 individual experiments performed in three technical replicates ; * * * , P = 0 . 0008 , mixed - effects analysis with Tukey ' s multiple comparisons test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Patient-derived and control fibroblasts were used. (A) FLAG-tagged tandem ubiquitin binding entity (TUBE) assay was performed to pull down linear ubiquitin linkages upon stimulation with 500ng/ml TNF. (B) The TNFR1-SC was isolated using 500ng/ml TAP-TNF. (C) Cells were stimulated with 100ng/ml TNF for the indicated times and analyzed by western blot. One representative (A - C) of three independent experiments is shown.(E) B cells were incubated with 50µg/ml cycloheximide (CHX) for the indicated times and subjected to analysis by western blot. One representative (left panel) out of 4 individual experiments analyzed by densitometry (right panel) is shown. Data are presented as mean ± SD. *, P = 0.02, unpaired t-test.(G) Fibroblasts were incubated with 50µg/ml cycloheximide (CHX) or DMSO and stimulated with TNF as indicated. Viability was measured after 24 hours. Data are presented as mean ± SD of 5 individual experiments performed in three technical replicates; ***, P = 0.0008, mixed-effects analysis with Tukey's multiple comparisons test.