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 .

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

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 .

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

F. 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 .

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

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 .

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

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

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.


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 .

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

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 ) .

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

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 .

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

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 .

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

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.


D . HEK293 cells were treated for 8 h with 20 μM

D. HEK293 cells were treated for 8 h with 20 μM neocarzinostatin


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 .

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

E. 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O

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.


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 .

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

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 .

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

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

C. 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.


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 .

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

A, B. 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 .

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

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 .

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

D. 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 .

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

E. 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 .

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

F. 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 .

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

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

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.


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 .

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

A, B. 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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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

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.


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 .

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

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 .

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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 .

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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 .

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

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 .

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

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 .

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

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.


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 .

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

A, B. 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 .

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

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 .

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

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 .

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

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 .

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

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

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.


( 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 ) .

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

(C) 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 .

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(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.


( 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 .

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

(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 O O O O O O O O O O O O O O O O O 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) 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.


Biochemical 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 .

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

Biochemical 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 .

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

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 .

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(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.


( 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 .

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(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.


Patient - 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 .

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Patient-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 .

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(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 .

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(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.


Paneth cell quantification on Lgr5 - DTReGFP duodenum ( Duo ) and Ileum ( Ile ) . Left panel : Representative images of Lendrum ' s staining that evidence Paneth cell granules . Arrows show differentiated Paneth cells . Right panel : quantification of the Paneth cells per 10 intervilli regions . Each symbol indicates the value for a given embryo .

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Paneth cell quantification on Lgr5-DTReGFP duodenum (Duo) and Ileum (Ile). Left panel: Representative images of Lendrum's staining that evidence Paneth cell granules. Arrows show differentiated Paneth cells. Right panel: quantification of the Paneth cells per 10 intervilli regions. Each symbol indicates the value for a given embryo.


Expression analysis by qRT - PCR of the indicated Paneth cell markers in Lgr5 - DTReGFP ileums ( n = 8 WT , 12 HE , 10 KO ) .

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Expression analysis by qRT-PCR of the indicated Paneth cell markers in Lgr5-DTReGFP ileums (n= 8 WT, 12 HE, 10 KO).


X - gal staining in Axin2Lac / + - Lgr5 - GFP - CreERT2 WT or KO ileums .

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X-gal staining in Axin2Lac/+-Lgr5-GFP-CreERT2 WT or KO ileums.


Gene expression analysis by qRT - PCR of stem cell markers in Lgr5 - DTReGFP ileums ( n = 3 WT , 6 HE , 6 KO ) .

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Gene expression analysis by qRT-PCR of stem cell markers in Lgr5-DTReGFP ileums (n= 3 WT, 6 HE, 6 KO).


FACS quantification of the number of eGFP + ve ( ISC ) cells per small intestine at the indicated developmental stages in Lgr5 HEs and Lgr5 KOs . Each symbol indicates the value for a given embryo . Mean value for each group is depicted on the graph .

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FACS quantification of the number of eGFP+ve (ISC) cells per small intestine at the indicated developmental stages in Lgr5 HEs and Lgr5 KOs. Each symbol indicates the value for a given embryo. Mean value for each group is depicted on the graph.


Plating efficiency of ex vivo cultured E18 . 5 small intestines quantified 6 days after initial seeding . Each symbol indicates the value for a given embryo .

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Plating efficiency of ex vivo cultured E18.5 small intestines quantified 6 days after initial seeding. Each symbol indicates the value for a given embryo.


Influence of mouse Rspondin 1 concentration on growth of replated Lgr5 - DTReGFP WT and Lgr5 KO organoids after 5 days of culture .

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Influence of mouse Rspondin 1 concentration on growth of replated Lgr5-DTReGFP WT and Lgr5 KO organoids after 5 days of culture.


Paneth cell quantification on Lgr5 - DTReGFP ileums . Each symbol indicates the value for a given embryo .

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Paneth cell quantification on Lgr5-DTReGFP ileums. Each symbol indicates the value for a given embryo.


Immunofluorescence showing Olfm4 + ve cells in Lgr5 - DTReGFP duodenums . Cell membranes are shown with b - catenin and nuclei were counterstained with DAPI . Quantification of Olfm4 + ve cells per intervilli regions in duodenum and ileum . Each symbol indicates the value for a given embryo .

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Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP duodenums. Cell membranes are shown with b-catenin and nuclei were counterstained with DAPI. Quantification of Olfm4+ve cells per intervilli regions in duodenum and ileum. Each symbol indicates the value for a given embryo.


Axin2 expression levels detected by RNAscope on Lgr5 - DTReGFP proximal intestine .

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Axin2 expression levels detected by RNAscope on Lgr5-DTReGFP proximal intestine.


Gene expression analysis by qRT - PCR of stem cell and differentiation markers in Lgr5 - DTReGFP ileums ( vehicle - treated : 7 WT and 7 KO ; LGK974 - treated : 3 WT and 7 KO ) .

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Gene expression analysis by qRT-PCR of stem cell and differentiation markers in Lgr5-DTReGFP ileums (vehicle-treated: 7 WT and 7 KO; LGK974-treated: 3 WT and 7 KO).


Representative pictures of lineage tracing in Lgr5 - expressing or Lgr5 cKO intestine after 10 days of chase and quantification of the number of traced clones per recombined surface at PN18 ( ileum ) . Each symbol indicates the value for a given mouse .

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Representative pictures of lineage tracing in Lgr5-expressing or Lgr5 cKO intestine after 10 days of chase and quantification of the number of traced clones per recombined surface at PN18 (ileum). Each symbol indicates the value for a given mouse.


Representative immunofluorescence pictures showing Paneth cells ( Plyz + ve ) in traced clones ( RFP + ve ) from control ( Lgr5Cre / + ) and cKO ( Lgr5Cre / flox ) PN18 and adult ileums . Arrow heads point to Plyz + ve cells in traced clones . Quantification of the number of Paneth cell number per recombined RFP + ve crypt on control and cKO ileums in PN18 . Each symbol indicates the value for a given mouse .

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Representative immunofluorescence pictures showing Paneth cells (Plyz+ve) in traced clones (RFP+ve) from control (Lgr5Cre/+) and cKO (Lgr5Cre/flox) PN18 and adult ileums. Arrow heads point to Plyz+ve cells in traced clones. Quantification of the number of Paneth cell number per recombined RFP+ve crypt on control and cKO ileums in PN18. Each symbol indicates the value for a given mouse.


ISC ( eGFP + ve ) cells were FACS - sorted from Lgr5 - DTReGFP embryos at E16 . 5 and subjected to RNAseq analysis . Heatmap of differentially regulated genes in HE and KO ISCs at E16 . 5 . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO )

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ISC (eGFP+ve) cells were FACS-sorted from Lgr5-DTReGFP embryos at E16.5 and subjected to RNAseq analysis. Heatmap of differentially regulated genes in HE and KO ISCs at E16.5. Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO)


Compared Hallmarks for upregulated and downregulated genes in the transcriptome of E16 . 5 Lgr5 KOs vs E16 . 5 HEs , adult HEs vs E18 . 5 HE embryos and adult Lgr5 - Cre cKO vs HEs . * Asterisks refer to given pathways for which differentially modulated genes were found upregulated and downregulated . Right panels : GSEA showing enrichment of the epithelial to mesenchymal transition ( EMT ) data set in E16 . 5 Lgr5 KOs vs E16 . 5 HEs , adult HEs vs E18 . 5 HE embryos , and adult Lgr5 cKO vs adult HE modulated genes . NES : Normalized Enrichment Score . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO ) ; Lgr5 - DTReGFP E18 . 5 individual embryos ( n = 2 HE ) and Lgr5 - DTReGFP adult individual animals ( n = 2 HE ) ; Lgr5 - Cre / flox adult individual animals ( n = 2 HE , 3 cKO ) .

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Compared Hallmarks for upregulated and downregulated genes in the transcriptome of E16.5 Lgr5 KOs vs E16.5 HEs, adult HEs vs E18.5 HE embryos and adult Lgr5-Cre cKO vs HEs. * Asterisks refer to given pathways for which differentially modulated genes were found upregulated and downregulated. Right panels: GSEA showing enrichment of the epithelial to mesenchymal transition (EMT) data set in E16.5 Lgr5 KOs vs E16.5 HEs, adult HEs vs E18.5 HE embryos, and adult Lgr5 cKO vs adult HE modulated genes. NES: Normalized Enrichment Score. Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO); Lgr5-DTReGFP E18.5 individual embryos (n=2 HE) and Lgr5-DTReGFP adult individual animals (n=2 HE); Lgr5-Cre/flox adult individual animals (n= 2 HE, 3 cKO).


Expression levels of genes commonly downregulated in ISCs of Lgr5 KOs vs Lgr5 HEs at E16 . 5 and Lgr5 adult vs E18 . 5 HEs . RPKM : reads per kilobase of transcript per million mapped reads . Below : GeneOntology analysis of downregulated genes identified in the Epithelial Mesenchymal Transition Gene Set ( Hallmark MSigDB collection ) . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO ) ; Lgr5 - DTReGFP E18 . 5 individual embryos ( n = 2 HE ) and Lgr5 - DTReGFP adult individual animals ( n = 2 HE ) ; Lgr5 - Cre / flox adult individual animals ( n = 2 HE , 3 cKO ) . Data are represented as means ± sem ( C ) .

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Expression levels of genes commonly downregulated in ISCs of Lgr5 KOs vs Lgr5 HEs at E16.5 and Lgr5 adult vs E18.5 HEs. RPKM: reads per kilobase of transcript per million mapped reads. Below: GeneOntology analysis of downregulated genes identified in the Epithelial Mesenchymal Transition Gene Set (Hallmark MSigDB collection). Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO); Lgr5-DTReGFP E18.5 individual embryos (n=2 HE) and Lgr5-DTReGFP adult individual animals (n=2 HE); Lgr5-Cre/flox adult individual animals (n= 2 HE, 3 cKO). Data are represented as means ± sem (C).


Expression profile of Col1a1 , Zeb2 and Cxcl10 genes detected by RNAscope in embryonic and adult intestines . Insets with higher magnification are depicted on the right of each corresponding picture , boundaries between epithelium and mesenchyme are shown with dotted lines and arrows point to epithelial expression .

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Expression profile of Col1a1, Zeb2 and Cxcl10 genes detected by RNAscope in embryonic and adult intestines. Insets with higher magnification are depicted on the right of each corresponding picture, boundaries between epithelium and mesenchyme are shown with dotted lines and arrows point to epithelial expression.


Expression of Axin2 , Lgr5 , Lgr4 , Rspo1 , Rspo2 , Rspo3 and Rspo4 genes was analyzed on E15 , E16 . 5 and adult stages by RNAscope . Arrows evidence expressing cells .

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Expression of Axin2, Lgr5, Lgr4, Rspo1, Rspo2, Rspo3 and Rspo4 genes was analyzed on E15, E16.5 and adult stages by RNAscope. Arrows evidence expressing cells.


Representative pictures showing the influence of mouse Rspondin type and concentration on growth of replated Lgr5 - DTReGFP WT and Lgr5 KO organoids after 4 days of culture . Arrows show alive elements and arrow heads dead elements .

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Representative pictures showing the influence of mouse Rspondin type and concentration on growth of replated Lgr5-DTReGFP WT and Lgr5 KO organoids after 4 days of culture. Arrows show alive elements and arrow heads dead elements.


Organoid morphology depends on Rspondin ligand type and concentration . Left panel : growth quantified as the percentage of elements present at day1 surviving at day4 . A mean of 100 elements were analyzed per sample and per condition ( n : 4 WT , 2 KO organoid culture samples , each originating from a given embryo ) . Right panel : organoid complexity measured by branching coefficient . Each symbol indicates the value of a given organoid ( analysis was done on 4 WT and 2 KO organoid culture samples , each originating from a given embryo ) .

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Organoid morphology depends on Rspondin ligand type and concentration. Left panel: growth quantified as the percentage of elements present at day1 surviving at day4. A mean of 100 elements were analyzed per sample and per condition (n: 4 WT, 2 KO organoid culture samples, each originating from a given embryo). Right panel: organoid complexity measured by branching coefficient. Each symbol indicates the value of a given organoid (analysis was done on 4 WT and 2 KO organoid culture samples, each originating from a given embryo).


Heatmap showing the impact of Rspondin type and concentration ( ng / ml ) on relative Wnt target gene expression ( list from the Sansom APC data set ) in Lgr5 - DTReGFP WT and KO organoids analyzed by RNAseq ( n = 3 WT and 2 organoid culture samples , each originating from a given embryo ) .

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Heatmap showing the impact of Rspondin type and concentration (ng/ml) on relative Wnt target gene expression (list from the Sansom APC data set) in Lgr5-DTReGFP WT and KO organoids analyzed by RNAseq (n= 3 WT and 2 organoid culture samples, each originating from a given embryo).


Gene expression analysis by qRT - PCR of stem cell markers in Lgr5 - DTREeGFP WT and KO organoids after 5 days of culture ( expression relative to reference genes ) . Each symbol indicates the value for an organoid culture originated from a given embryo .

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Gene expression analysis by qRT-PCR of stem cell markers in Lgr5-DTREeGFP WT and KO organoids after 5 days of culture (expression relative to reference genes). Each symbol indicates the value for an organoid culture originated from a given embryo.


Immunofluorescence showing Olfm4 + ve cells in Lgr5 - DTReGFP WT and KO organoids cultured in Rspo2 conditions . Epithelium is delineated with b - catenin and nuclei counterstained with DAPI ( Merge ) . Insets : white arrowheads evidence Olfm4 + ve cells concentrated in organoid protrusions . Quantification of Olfm4 + ve area with respect to the total epithelial area . Each symbol indicates the value for an organoid culture originating from a given embryo .

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Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP WT and KO organoids cultured in Rspo2 conditions. Epithelium is delineated with b-catenin and nuclei counterstained with DAPI (Merge). Insets: white arrowheads evidence Olfm4+ve cells concentrated in organoid protrusions. Quantification of Olfm4+ve area with respect to the total epithelial area. Each symbol indicates the value for an organoid culture originating from a given embryo.


( A ) PI3Kγ is expressed by human hepatocytes and infiltrating immune cells in biopsies from patients with minimal to mild inflammatory activity . Triangles point to immune cells ( clusters ) , including some neutrophils known to express PI3Kγ highly . In the negative control , the primary antibody was replaced by an equal volume buffer . The number of included patients , gender , and diagnosis are summarized

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(A) PI3Kγ is expressed by human hepatocytes and infiltrating immune cells in biopsies from patients with minimal to mild inflammatory activity. Triangles point to immune cells (clusters), including some neutrophils known to express PI3Kγ highly. In the negative control, the primary antibody was replaced by an equal volume buffer. The number of included patients, gender, and diagnosis are summarized


( A ) PI3Kγ is expressed by human hepatocytes and infiltrating immune cells in biopsies from patients with minimal to mild inflammatory activity . Triangles point to immune cells ( clusters ) , including some neutrophils known to express PI3Kγ highly . In the negative control , the primary antibody was replaced by an equal volume buffer . The number of included patients , gender , and diagnosis are

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( B ) PI3Kγ expression in human primary hepatocytes from 20 male ( ♂ ) or female ( ♀ ) donor pools ( HEP , DP20 ) , but not non - parenchymal cells ( NPC ) . Isolated human leukocytes from healthy volunteers ( LEU ) served as positive controls . The characterization of the donor pools is summarized

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(B) PI3Kγ expression in human primary hepatocytes from 20 male (♂) or female (♀) donor pools (HEP, DP20), but not non-parenchymal cells (NPC). Isolated human leukocytes from healthy volunteers (LEU) served as positive controls. The characterization of the donor pools is summarized


( B ) PI3Kγ expression in human primary hepatocytes from 20 male ( ♂ ) or female ( ♀ ) donor pools ( HEP , DP20 ) , but not non - parenchymal cells ( NPC ) . Isolated human leukocytes from healthy volunteers ( LEU ) served as positive controls . The characterization of the donor pools is

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( D ) Survival of WT , PI3Kγ null ( left ) and liver - specific PI3Kγ knockout mice ( PI3Kγ floxtg / tg x AlbCre ( tg ) / tg , middle ) or systemic application of the PI3Kγ inhibitor , AS605240 ( right ) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension . Animal numbers : B6 : 86 ( % male : 52 , % female : 48 ) , B6 - Pi3kcgko / ko : 59 ( % male : 51 , % female 49 ) , B6 - Pi3kcgflox / flox x AlbCre ( tg ) / tg : 25 ( % male : 68 , % female 32 ) , FVB / N Vehicle : 25 ( % male : 44 , % female 56 ) , FVB / N AS : 43 ( % male : 44 , % female 56 )

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(D) Survival of WT, PI3Kγ null (left) and liver-specific PI3Kγ knockout mice (PI3Kγ floxtg/tg x AlbCre(tg)/tg, middle) or systemic application of the PI3Kγ inhibitor, AS605240 (right) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension. Animal numbers: B6: 86 (%male: 52, %female: 48), B6-Pi3kcgko/ko: 59 (%male: 51, %female 49), B6-Pi3kcgflox/flox x AlbCre(tg)/tg: 25 (%male: 68, %female 32), FVB/N Vehicle: 25 (%male: 44, %female 56), FVB/N AS: 43 (%male: 44, %female 56)


( C ) Uptake characteristics of T - LipoAS by primary human hepatocytes demonstrate competitive inhibition by cyclosporin A - and energy dependence , confirming active uptake by organic anion transporters ( OATPs ) . The data points are depicted mean ± SD from two batches of primary human hepatocytes in quadruples .

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(C) Uptake characteristics of T-LipoAS by primary human hepatocytes demonstrate competitive inhibition by cyclosporin A- and energy dependence, confirming active uptake by organic anion transporters (OATPs). The data points are depicted mean ± SD from two batches of primary human hepatocytes in quadruples.


( F ) T - LipoAS effectively inhibits fMLP - stimulated PI3Kγ , reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes . The mean pAKT / AKT ratio and standard deviation ( SD ) were calculated for all experiments separately .

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(F) T-LipoAS effectively inhibits fMLP-stimulated PI3Kγ, reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes. The mean pAKT/AKT ratio and standard deviation (SD) were calculated for all experiments separately.


( G ) Episcopic imaging of mice treated with T - LipoAS for 15 or 60 min and relative concentration of the targeting moiety DY - 635 in the different compartments .

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(G) Episcopic imaging of mice treated with T-LipoAS for 15 or 60 min and relative concentration of the targeting moiety DY-635 in the different compartments.


( B Pro - anti - inflammatory cytokines were analyzed in EDTA - plasma from peritoneal contamination and infection ( PCI ) and sham animals . # p < 0 . 05 against sham , * p < 0 . 05 as indicated ; Kruskal - Wallis ANOVA with controlled false - discovery rates ( Benjamini - Hochberg procedure ) .

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(B Pro- anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).


C ) anti - inflammatory cytokines were analyzed in EDTA - plasma from peritoneal contamination and infection ( PCI ) and sham animals . # p < 0 . 05 against sham , * p < 0 . 05 as indicated ; Kruskal - Wallis ANOVA with controlled false - discovery rates ( Benjamini - Hochberg procedure ) .

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C) anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).


( F , G ) Concentration of ( F ) neutrophils and ( G ) monocytes in 2 mL peritoneal lavage .

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(F,G) Concentration of (F) neutrophils and (G) monocytes in 2 mL peritoneal lavage.


( A ) Intravital microscopy of elimination of CDFDA , a fluorescent substrate subject to excretory elimination by hepatocytes via Mrp - 2 , allows direct visualization of excretory function in sham or septic ( PCI ) animals treated with vehicle or a hepatocyte - directed inhibitor of PI3Kγ ( T - LipoAS ) .

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(A) Intravital microscopy of elimination of CDFDA, a fluorescent substrate subject to excretory elimination by hepatocytes via Mrp-2, allows direct visualization of excretory function in sham or septic (PCI) animals treated with vehicle or a hepatocyte-directed inhibitor of PI3Kγ (T-LipoAS).


a - e , Scanning electron microscopy ( SEM ) images of fly eyes expressing DTS7 with or without the indicated HDAC6 transgenes . Lower panels , magnification of ommatidia . a , Normal eyes in DTS7 flies reared at 22 ° C . b , Rough eyes in DTS7 flies reared at 28 ° C . c , d , Degeneration was suppressed by expression of Drosophila HDAC6 ( dHDAC6 ; c ) or human HDAC6 ( hHDAC6 ; d ) , but not a catalytically dead mutant of hHDAC6 ( cat . mut . ; e ) .

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

a-e, Scanning electron microscopy (SEM) images of fly eyes expressing DTS7 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. a, Normal eyes in DTS7 flies reared at 22 °C. b, Rough eyes in DTS7 flies reared at 28 °C. c, d, Degeneration was suppressed by expression of Drosophila HDAC6 (dHDAC6; c) or human HDAC6 (hHDAC6; d), but not a catalytically dead mutant of hHDAC6 (cat. mut.; e).


f - j , SEM images of fly eyes expressing AR52 with or without the indicated HDAC6 transgenes . Lower panels , magnification of ommatidia . f , Normal eyes in AR52 flies reared without ( - ) DHT . g , Rough eyes in AR52 flies reared with ( + ) DHT . Degeneration was suppressed by expression of Drosophila HDAC6 ( h ) or human HDAC6 ( i ) , but not a catalytically dead mutant of human HDAC6 ( j ) .

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f-j, SEM images of fly eyes expressing AR52 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. f, Normal eyes in AR52 flies reared without (-) DHT. g, Rough eyes in AR52 flies reared with (+) DHT. Degeneration was suppressed by expression of Drosophila HDAC6 (h) or human HDAC6 (i), but not a catalytically dead mutant of human HDAC6 (j).


k - p , Detection of UPS reporter in imaginal eye discs from third - instar larvae by confocal microscopy . High level fluorescence was found in flies expressing GFP ( k , positive control ) , but fluorescence was barely detectable in control flies expressing CL1 - GFP ( l , negative control ) . CL1 - GFP accumulates in DTS7flies with temperature - dependent proteasome impairment ( compare m to n ) and in AR52flies with ligand - dependent degeneration ( compare o to p ) . The retinal phenotypes of 200 to > 1 , 000 flies of each genotype were examined . Quantitative analyses of eye phenotypes and proteasome impairment are presented in Supplementary Figs S2 and S3 , respectively . ( DHT , dihydrotestosterone . )

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

k-p, Detection of UPS reporter in imaginal eye discs from third-instar larvae by confocal microscopy. High level fluorescence was found in flies expressing GFP (k, positive control), but fluorescence was barely detectable in control flies expressing CL1-GFP (l, negative control). CL1-GFP accumulates in DTS7flies with temperature-dependent proteasome impairment (compare m to n) and in AR52flies with ligand-dependent degeneration (compare o to p). The retinal phenotypes of 200 to >1,000 flies of each genotype were examined. Quantitative analyses of eye phenotypes and proteasome impairment are presented in Supplementary Figs S2 and S3, respectively. (DHT, dihydrotestosterone.)


a - e , Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in ( f ) . a , An autophagosome ( red arrow ) containing cytoplasmic contents in a photoreceptor neuron from an AR52fly reared on DHT . b , Higher magnification of the autophagosome in a . c , Multiple autophagolysosomes ( red arrows ) containing dense , amorphous material from an AR52fly reared on DHT . d , A juxtanuclear multilamellar body ( red arrow ) from a DTS7fly reared at 28 ° C . e , A multivesicular body ( red arrow ) from a DTS7fly reared at 28 ° C . f , A significant increase in the frequency of neurons with autophagic figures in DTS7 flies reared at 28 ° C compared to those reared at 22 ° C , and in AR52 flies reared on DHT compared to those reared off DHT . Data show mean ± s . d . , n = 59 - 82 neurons in 5 sections per condition . No accumulation of autophagic figures was found in AR12 flies .

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

a-e, Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in (f). a, An autophagosome (red arrow) containing cytoplasmic contents in a photoreceptor neuron from an AR52fly reared on DHT. b, Higher magnification of the autophagosome in a. c, Multiple autophagolysosomes (red arrows) containing dense, amorphous material from an AR52fly reared on DHT. d, A juxtanuclear multilamellar body (red arrow) from a DTS7fly reared at 28 °C. e, A multivesicular body (red arrow) from a DTS7fly reared at 28 °C. f, A significant increase in the frequency of neurons with autophagic figures in DTS7 flies reared at 28 °C compared to those reared at 22 °C, and in AR52 flies reared on DHT compared to those reared off DHT. Data show mean ± s.d., n = 59-82 neurons in 5 sections per condition. No accumulation of autophagic figures was found in AR12 flies.


g - l , SEM images of fly eyes expressing the indicated transgenes . RNAi knockdown ( KD ) of atg6 and atg12 enhances degeneration in DTS7 flies reared at 28 ° C ( compare h , i to g ) and AR52 flies reared on DHT ( compare k , l to j ) . 200 to > 1 , 000 fly eyes of each genotype were examined . Quantitative analyses of eye phenotypes are presented in Supplementary Fig . S2 . ( N , nucleus ; Rh , rhabdomere . )

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

g-l, SEM images of fly eyes expressing the indicated transgenes. RNAi knockdown (KD) of atg6 and atg12 enhances degeneration in DTS7 flies reared at 28 °C (compare h, i to g) and AR52 flies reared on DHT (compare k, l to j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2. (N, nucleus; Rh, rhabdomere.)


a , b , d , Western blots from flies expressing the indicated transgenes . a , Steady state levels of AR52 protein are reduced in flies overexpressing Drosophila HDAC6 , but are elevated in flies in which atg6 or atg12 has been knocked down . b , Western blots showing the temporal profile of AR52 protein monomer and high molecular weight aggregate levels after a brief pulse of expression . AR52 protein became detectable by 2 . 5 h after treatment with RU486 , reached a peak at 10 h , and then slowly decayed . c , A logarithmic plot of AR52 / actin ratios was used to determine the line of best fit by regression analysis ( y = Ae - Kx ) . R2 = 0 . 9117 ( AR52 - DHT ) , R2 = 0 . 7808 ( AR52 + DHT ) , R2 = 0 . 9719 ( HDAC6 + ( AR52 - DHT ) ) , R2 = 0 . 9644 ( HDAC6 + ( AR52 + DHT ) ) . Half - life was determined by the slope of the best fit line with the equation t1 / 2 = 0 . 693 / K . Half - life of AR52 in vivo was reduced ∼ 2 - fold in flies co - expressing Drosophila HDAC6 and did not differ significantly in the presence ( broken lines ) or absence ( solid lines ) of DHT . Plots of the mean AR52 / actin ratios are shown in Supplementary Fig . S12 . d , Flies co - expressing Drosophila HDAC6 showed a nearly identical profile of induced expression as in b , but AR protein decayed at an accelerated rate . Exogenous Drosophila HDAC6 was detected by immunoblot against the V5 epitope .

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

a, b, d, Western blots from flies expressing the indicated transgenes. a, Steady state levels of AR52 protein are reduced in flies overexpressing Drosophila HDAC6, but are elevated in flies in which atg6 or atg12 has been knocked down. b, Western blots showing the temporal profile of AR52 protein monomer and high molecular weight aggregate levels after a brief pulse of expression. AR52 protein became detectable by 2.5 h after treatment with RU486, reached a peak at 10 h, and then slowly decayed. c, A logarithmic plot of AR52/actin ratios was used to determine the line of best fit by regression analysis (y = Ae-Kx). R2 = 0.9117 (AR52 - DHT), R2 = 0.7808 (AR52 + DHT), R2 = 0.9719 (HDAC6 + (AR52 - DHT)), R2 = 0.9644 (HDAC6 + (AR52 + DHT)). Half-life was determined by the slope of the best fit line with the equation t1/2 = 0.693/K. Half-life of AR52 in vivo was reduced ∼2-fold in flies co-expressing Drosophila HDAC6 and did not differ significantly in the presence (broken lines) or absence (solid lines) of DHT. Plots of the mean AR52/actin ratios are shown in Supplementary Fig. S12. d, Flies co-expressing Drosophila HDAC6 showed a nearly identical profile of induced expression as in b, but AR protein decayed at an accelerated rate. Exogenous Drosophila HDAC6 was detected by immunoblot against the V5 epitope.


a - l , SEM images of fly eyes expressing the indicated transgenes . The rough eye phenotypes caused by proteasome mutation ( a ) or by expression of polyQ - expanded AR ( b ) were both suppressed by rearing flies on the TOR inhibitor rapamycin ( c , d ) . e , f , Rapamycin failed to suppress degeneration in an autophagy - deficient background created by knockdown of atg12 , confirming that rescue by rapamycin is autophagy - dependent . g , h , Rapamycin also failed to suppress degeneration when HDAC6 levels were knocked down , demonstrating that suppression via the TOR pathway is HDAC6 - dependent . k , j , HDAC6 failed to suppress degeneration in an autophagy - deficient background , confirming that rescue by HDAC6 is dependent on autophagy ( compare k , l to i , j ) . 200 to > 1 , 000 fly eyes of each genotype were examined . Quantitative analyses of eye phenotypes are presented in Supplementary Fig . S2 .

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

a-l, SEM images of fly eyes expressing the indicated transgenes. The rough eye phenotypes caused by proteasome mutation (a) or by expression of polyQ-expanded AR (b) were both suppressed by rearing flies on the TOR inhibitor rapamycin (c, d). e, f, Rapamycin failed to suppress degeneration in an autophagy-deficient background created by knockdown of atg12, confirming that rescue by rapamycin is autophagy-dependent. g, h, Rapamycin also failed to suppress degeneration when HDAC6 levels were knocked down, demonstrating that suppression via the TOR pathway is HDAC6-dependent. k, j, HDAC6 failed to suppress degeneration in an autophagy-deficient background, confirming that rescue by HDAC6 is dependent on autophagy (compare k, l to i, j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2.


B . Survival frequencies observed after DSB induction at TEL6R in WT , dnl4Δ , sir4Δ cells , expressing or not high levels of Sir3p ( oeSir3 and WT respectively ) . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . C . Survival frequencies observed after DSB induction at LYS2 in the indicated strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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B. Survival frequencies observed after DSB induction at TEL6R in WT, dnl4Δ, sir4Δ cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Error bars indicate survival standard error (SEM) of at least three independent experiments. C. Survival frequencies observed after DSB induction at LYS2 in the indicated strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).


F . Representative images of Mre11 - YFP foci in response to an I - SceI - induced DSB at LYS2 in WT cells , expressing or not high levels of Sir3p ( oeSir3 and WT respectively ) . Scale bars are 2 μm . G . Quantification of cells with DSB induced Mre11 - YFP foci after DSB induction at LYS2 I - SceI cleavage site in WT , sae2Δ and Sir3 overexpressing ( oeSir3 ) strains . Error bars indicate survival standard error ( SEM ) of at least three independent experiments . Data information : Significance was determined using 2 - tailed , unpaired Student ' s t test . * P - value 0 . 01 to 0 . 05 , significant ; * * P - value 0 . 001 to 0 . 01 , very significant ; * * * P - value 0 . 0001 to 0 . 001 , extremely significant ; * * * * P < 0 . 0001 , extremely significant ; P ≥ 0 . 05 , not significant ( ns ) .

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F. Representative images of Mre11-YFP foci in response to an I-SceI-induced DSB at LYS2 in WT cells, expressing or not high levels of Sir3p (oeSir3 and WT respectively). Scale bars are 2 μm. G. Quantification of cells with DSB induced Mre11-YFP foci after DSB induction at LYS2 I-SceI cleavage site in WT, sae2Δ and Sir3 overexpressing (oeSir3) strains. Error bars indicate survival standard error (SEM) of at least three independent experiments. Data information: Significance was determined using 2-tailed, unpaired Student's t test. *P-value 0.01 to 0.05, significant; **P-value 0.001 to 0.01, very significant; ***P-value 0.0001 to 0.001, extremely significant; ****P < 0.0001, extremely significant; P ≥ 0.05, not significant (ns).