( F - I ) In vitro proteasome activity assays were performed as described in Materials and Methods . Bars represent means + S . D . from three independent experiments . For statistical analysis , student t - test ( two - sided , one type ) were used . * P value : ( F ) , 0 . 0039 ; ( G ) , 0 . 0052 ; ( H ) , 0 . 0009 , ( I ) DFC , 0 . 00032 ; ( I ) - HEK293 , 0 . 0039 ; ( I ) - HeLa : 0 . 00051 .

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(F-I) In vitro proteasome activity assays were performed as described in Materials and Methods. Bars represent means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: (F), 0.0039; (G), 0.0052; (H), 0.0009, (I) DFC, 0.00032; (I)-HEK293, 0.0039; (I)-HeLa: 0.00051.


( J ) XO8 GSCs and XO8 DFCs treated with MG132 as described in Materials and Methods were labeled with annexin V and visualized using fluorescent microscopy . Scale bar , 100 μM .

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(J) XO8 GSCs and XO8 DFCs treated with MG132 as described in Materials and Methods were labeled with annexin V and visualized using fluorescent microscopy. Scale bar, 100 μM.


( K ) Cells from ( J ) were subjected to flow cytometry analysis . Annexin V - positive cell numbers were obtained from three independent experiments and normalized to those of controls cells treated with DMSO . Bars , means + S . D . from three independent experiments . For statistical analysis , student t - test ( two - sided , one type ) were used . * P value : 6 hrs , 0 . 0012 ; 17 hrs , 0 . 0041 .

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(K) Cells from (J) were subjected to flow cytometry analysis. Annexin V-positive cell numbers were obtained from three independent experiments and normalized to those of controls cells treated with DMSO. Bars, means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: 6 hrs, 0.0012; 17 hrs, 0.0041.


( L ) Cells treated with 50 nM MG132 were subjected to immunoblotting analysis of apoptotic markers .

(L) Cells treated with 50 nM MG132 were subjected to immunoblotting analysis of apoptotic markers.


( M and N ) Similar to ( L ) except that XO6 and XO10 cells were used .

(M and N) Similar to (L) except that XO6 and XO10 cells were used.


( A ) Real - time PCR analysis of the proteasomal subunits PSMB5 , PSMB6 , and PSMB7 in XO8 GSCs and XO8 DFCs . Shown are means + S . D . values from the three independent experiments following normalization to those of GSCs . For statistical analysis , student t - test ( two - sided , one type ) were used . * * P value = PSMB5 : 0 . 053 , PSMB6 : 0 . 034 , PSMB7 : 0 . 067 . ( B ) Similar to ( A ) except that the cells were treated with 50 nM MG132 for 17 hrs . Shown are means + S . D . values from the three independent experiments following normalization to those of control cells treated with DMSO . For statistical analysis , student t - test ( two - sided , one type ) were used . * * P value = PSMB5 : 0 . 022 , PSMB6 : 0 . 08 , PSMB7 : 0 . 603 .

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(A) Real-time PCR analysis of the proteasomal subunits PSMB5, PSMB6, and PSMB7 in XO8 GSCs and XO8 DFCs. Shown are means +S.D. values from the three independent experiments following normalization to those of GSCs. For statistical analysis, student t-test (two-sided, one type) were used. ** P value=PSMB5: 0.053, PSMB6: 0.034, PSMB7: 0.067. (B) Similar to (A) except that the cells were treated with 50 nM MG132 for 17 hrs. Shown are means +S.D. values from the three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. ** P value=PSMB5: 0.022, PSMB6: 0.08, PSMB7: 0.603.


( C ) Immunoblotting analysis of proteasomal subunits in XO8 GSCs in comparison with XO8 DFCs at passages 11 and 12 . Note that part of Figure 5C is also shown as the left panel of Figure 4A . ( D ) Same as ( C ) except that the cells were treated with MG132 for 17 hrs .

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(C) Immunoblotting analysis of proteasomal subunits in XO8 GSCs in comparison with XO8 DFCs at passages 11 and 12. Note that part of Figure 5C is also shown as the left panel of Figure 4A. (D) Same as (C) except that the cells were treated with MG132 for 17 hrs.


( E ) Immunoblotting analysis of the NF - κB pathway in XO8 GSCs and XO8 DFCs .

(E) Immunoblotting analysis of the NF-κB pathway in XO8 GSCs and XO8 DFCs.


( F ) Fractionation analysis of NF - κB in XO8 GSCs and XO8 DFCs treated with 50 nM MG132 for 17 hrs . PARP1 and tubulin were used as markers for the nucleus and cytoplasm , respectively .

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(F) Fractionation analysis of NF-κB in XO8 GSCs and XO8 DFCs treated with 50 nM MG132 for 17 hrs. PARP1 and tubulin were used as markers for the nucleus and cytoplasm, respectively.


( G ) Immunoblotting analysis of JNK and p38 .

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(G) Immunoblotting analysis of JNK and p38.


( H ) Cell viability assay was performed as in Fig . 2 with cells treated with MG132 alone or in combination with SB203580 . Shown are means + S . D . values from three independent experiments following normalization to those of control cells treated with DMSO . For statistical analysis , student t - test ( two - sided , one type ) were used . * * P value = 0 . 724 , 0 . 502 , 0 . 439 for 0 . 5 , 1 . 0 and 5 . 0 μM SB203580 , respectively at 0 . 1 μM MG132 , and 0 . 760 , 0 . 683 , 0 . 498 for 0 . 5 , 1 . 0 and 5 . 0 μM SB203580 , respectively at 0 . 5 μM MG132 .

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(H) Cell viability assay was performed as in Fig. 2 with cells treated with MG132 alone or in combination with SB203580. Shown are means +S.D. values from three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. ** P value = 0.724, 0.502, 0.439 for 0.5, 1.0 and 5.0 μM SB203580, respectively at 0.1 μM MG132, and 0.760, 0.683, 0.498 for 0.5, 1.0 and 5.0 μM SB203580, respectively at 0.5 μM MG132.


( I ) Experimental conditions were similar to ( H ) except that SP600129 was used . Shown are means + S . D . values from three independent experiments following normalization to those of control cells treated with DMSO . For statistical analysis , student t - test ( two - sided , one type ) were used . * P value = 0 . 009 , 0 . 002 , 0 . 07 for 0 . 5 , 1 . 0 and 5 . 0 μM SP600129 , respectively at 0 . 1 μM MG132 , and 0 . 018 , 0 . 025 , 0 . 09 for 0 . 5 , 1 . 0 and 5 . 0 μM SP600129 , respectively at 0 . 5 μM MG132 .

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(I) Experimental conditions were similar to (H) except that SP600129 was used. Shown are means +S.D. values from three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. * P value = 0.009, 0.002, 0.07 for 0.5, 1.0 and 5.0 μM SP600129, respectively at 0.1 μM MG132, and 0.018, 0.025, 0.09 for 0.5, 1.0 and 5.0 μM SP600129, respectively at 0.5 μM MG132.


( J ) Immunoblotting analysis of XO8 GSCs treated with 50 nM MG132 alone or in combination with the JNK inhibitor SP600129 .

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(J) Immunoblotting analysis of XO8 GSCs treated with 50 nM MG132 alone or in combination with the JNK inhibitor SP600129.


( A and B ) XO8 GSCs and XO8 DFCs were treated with 50 nM MG132 for indicated hours . The expression of genes was assessed by real - time PCR using total RNA extracted from MG132 - treated cells . Mean + S . D . values from the three independent experiments are shown after normalization to that of control cells treated with DMSO .

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(A and B) XO8 GSCs and XO8 DFCs were treated with 50 nM MG132 for indicated hours. The expression of genes was assessed by real-time PCR using total RNA extracted from MG132-treated cells. Mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO.


( C ) XO8 GSCs and XO8 DFCs treated with 50 nM MG132 as described in ( A ) were subjected to immunoblotting .

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(C) XO8 GSCs and XO8 DFCs treated with 50 nM MG132 as described in (A) were subjected to immunoblotting.


( D ) XO8 GSCs were treated with MG132 , salinosporamide A , epoxomicin , or bortezomib for 24 hrs . Cell lysates were subjected to immunoblot analysis with anti - PARP - 1 , anti - CHOP , anti - ATF3 , anti - ATF3 , and anti - BiP antibodies .

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(D) XO8 GSCs were treated with MG132, salinosporamide A, epoxomicin, or bortezomib for 24 hrs. Cell lysates were subjected to immunoblot analysis with anti-PARP-1, anti-CHOP, anti-ATF3, anti-ATF3, and anti-BiP antibodies.


( E ) XO8 cells were treated with 50 nM MG132 in the absence or presence of 5μM actinomycin D ( ActD ) for 6 h . The level of ATF3 mRNA was measured using semi - quantitative real - time PCR in comparison with β - actin .

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(E) XO8 cells were treated with 50 nM MG132 in the absence or presence of 5μM actinomycin D (ActD) for 6 h. The level of ATF3 mRNA was measured using semi-quantitative real-time PCR in comparison with β-actin.


( A ) HEK293 cells were transfected with ATF3 shRNA or vector ( pLKO1 ) , treated with 50 nM MG132 , and then subjected to immunoblotting .

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(A) HEK293 cells were transfected with ATF3 shRNA or vector (pLKO1), treated with 50 nM MG132, and then subjected to immunoblotting.


( B ) Individualized XO8 GSCs were transduced with lentiviral shRNAs against ATF3 or control as described in Materials and Methods . After four days , individualized GSCs were treated with 50 nM MG132 , and subjected to immunoblotting .

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(B) Individualized XO8 GSCs were transduced with lentiviral shRNAs against ATF3 or control as described in Materials and Methods. After four days, individualized GSCs were treated with 50 nM MG132, and subjected to immunoblotting.


( C ) XO8 GSCs were transfected with control or CHOP siRNA for 48 hrs and then treated with 50 nM MG132 for 24 hrs . Cell viability was determined using the Trypan blue exclusion assay . Error bars represent the mean ± SD from three independent experiments ( * P < 0 . 05 , n = 3 ) . For statistical analysis , student t - test ( two - sided , one type ) were used .

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(C) XO8 GSCs were transfected with control or CHOP siRNA for 48 hrs and then treated with 50 nM MG132 for 24 hrs. Cell viability was determined using the Trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments (*P < 0.05, n=3). For statistical analysis, student t-test (two-sided, one type) were used.


( D ) Immunoblotting analysis of ( C ) . The cells were collected , lysed , and subjected to immunoblot analysis with indicated antibodies .

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(D) Immunoblotting analysis of (C). The cells were collected, lysed, and subjected to immunoblot analysis with indicated antibodies.


( E ) Wild - type and CHOP - / - MEF cells were treated with MG132 for 24 hrs . Cell viability was determined using trypan blue exclusion assay . Error bars represent the mean ± SD from three separate experiments ( * P < 0 . 05 , n = 3 ) . For statistical analysis , student t - test ( two - sided , one type ) were used .

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(E) Wild-type and CHOP-/- MEF cells were treated with MG132 for 24 hrs. Cell viability was determined using trypan blue exclusion assay. Error bars represent the mean ± SD from three separate experiments (*P < 0.05, n=3). For statistical analysis, student t-test (two-sided, one type) were used.


( F ) Cell lysates obtained in ( E ) were subjected to immunoblot analysis with anti - PARP - 1 , anti - CHOP or anti - ATF3 antibody .

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(F) Cell lysates obtained in (E) were subjected to immunoblot analysis with anti-PARP-1, anti-CHOP or anti-ATF3 antibody.


( G ) The expression of NOXA was assessed with real - time PCR using total RNA extracted from XO8 GSCs treated with 50 nM MG132 alone or in combination with each inhibitor of ER stress - driven apoptosis ( salubrinal ; 15 µM , STK064652 ; 20 µM , STK047915 ; 20 µM ) . The mean + S . D . values from the three independent experiments are shown after normalization to that of control cells treated with DMSO . For statistical analysis , student t - test ( two - sided , one type ) was used . * P value : salubrinal , 0 . 0003 ; STK064650 , 0 . 0036 ; STK047915 , 0 . 0019 .

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(G) The expression of NOXA was assessed with real-time PCR using total RNA extracted from XO8 GSCs treated with 50 nM MG132 alone or in combination with each inhibitor of ER stress-driven apoptosis (salubrinal; 15 µM, STK064652; 20 µM, STK047915; 20 µM). The mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) was used. * P value: salubrinal, 0.0003; STK064650, 0.0036; STK047915, 0.0019.


( H ) Similar to ( G ) except that XO8 GSC transduced with lentiviral shRNAs were treated with MG132 . The mean + S . D . values from the three independent experiments are shown after normalization to that of control cells treated with DMSO . For statistical analysis , student t - test ( two - sided , one type ) was used . * P value : # 1 , 0 . 026 ; # 5 , 0 . 0033 ; # 6 , 0 . 0482 .

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(H) Similar to (G) except that XO8 GSC transduced with lentiviral shRNAs were treated with MG132. The mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) was used. * P value: #1, 0.026; #5, 0.0033; #6, 0.0482.


( I ) XO8 GSCs were treated with 20 nM MG132 alone or in combination with each inhibitor as described in ( C ) , and subjected to immunoblotting . Sal . : salubrinal .

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(I) XO8 GSCs were treated with 20 nM MG132 alone or in combination with each inhibitor as described in (C), and subjected to immunoblotting. Sal.: salubrinal.


( A and B ) XO8 GSCs were subcutaneously ( s . c . ) injected in the flank of mice . When the tumor volume reached approximately 180 mm3 , GSC - bearing mice were administered intraperitoneally ( i . p . ) with 1 mg / kg MG132 and 10 mg / kg STK alone or in combination on every day for 2 weeks . Tumors were dissected from the subcutaneous regions of nude mice . Inset picture shows the tumor in respective group . Therapeutic effects were monitored using bioluminescence imaging . ( C ) The bar diagram represents the tumor volumes . ( D ) Tumor weights were determined at the end of the study . ( E ) The tumor sizes in each group were determined by histological analysis at day 21 after tumor inoculation .

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(A and B) XO8 GSCs were subcutaneously (s.c.) injected in the flank of mice. When the tumor volume reached approximately 180 mm3, GSC-bearing mice were administered intraperitoneally (i.p.) with 1 mg/kg MG132 and 10 mg/kg STK alone or in combination on every day for 2 weeks. Tumors were dissected from the subcutaneous regions of nude mice. Inset picture shows the tumor in respective group. Therapeutic effects were monitored using bioluminescence imaging. (C) The bar diagram represents the tumor volumes. (D) Tumor weights were determined at the end of the study. (E) The tumor sizes in each group were determined by histological analysis at day 21 after tumor inoculation.


( F ) XO10 GSC were orthotopically injected into the brains of BALB / c nude mice . STK047915 , MG132 , or both were injected intraperitoneally to GSCs - bearing mice at day 14 after XO10 ( 5 × 105 cells ) inoculation . The PBS was used as a control . Representative photographs of H & amp ; amp ; E staining from each group show tumor growth . Magnification , × 1 .

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(F) XO10 GSC were orthotopically injected into the brains of BALB/c nude mice. STK047915, MG132, or both were injected intraperitoneally to GSCs-bearing mice at day 14 after XO10 (5×105 cells) inoculation. The PBS was used as a control. Representative photographs of H&amp;E staining from each group show tumor growth. Magnification, ×1.


A . HEK293 cells were treated for 8 and 24 h with 100 μM H2O2 . GSNOR was evaluated by Western blot . Densitometry of each lane is normalized to GADPH , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n ≥ 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 calculated with regard to H2O2 - untreated cells .

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A. HEK293 cells were treated for 8 and 24 h with 100 μM H2O2. GSNOR was evaluated by Western blot. Densitometry of each lane is normalized to GADPH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n ≥ 3 independent experiments. *, p<0.05; **, p<0.01 calculated with regard to H2O2-untreated cells.


B . HEK293 cells were treated for 8 h with 100 or 200 μM H2O2 . Nrf2 ( left panel ) and GSNOR ( right panel ) were evaluated by Western blot performed in nuclear and cytosol - enriched fractions . GAPDH and Lamin A / C were used as loading and purity controls of cytosol and nuclei , respectively . Densitometry of GSNOR immunoreactive bands is normalized to GADPH and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 .

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B. HEK293 cells were treated for 8 h with 100 or 200 μM H2O2. Nrf2 (left panel) and GSNOR (right panel) were evaluated by Western blot performed in nuclear and cytosol-enriched fractions. GAPDH and Lamin A/C were used as loading and purity controls of cytosol and nuclei, respectively. Densitometry of GSNOR immunoreactive bands is normalized to GADPH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.


C . HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of the Nrf2 inhibitor trigonelline ( Trig ) . Heme oxygenase 1 ( HMOX - 1 ) , glutamate : cysteine ligase ( GCL ) and GSNOR expressions were evaluated RT - qPCR analyses Results shown are the means ± SEM of n = 3 experiments run in triplicate . * p < 0 . 05 ; n . s . , not significant .

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C. HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of the Nrf2 inhibitor trigonelline (Trig). Heme oxygenase 1 (HMOX-1), glutamate:cysteine ligase (GCL) and GSNOR expressions were evaluated RT-qPCR analyses Results shown are the means ± SEM of n = 3 experiments run in triplicate. *p<0.05; n.s., not significant.


D . RT - qPCR analyses of GSNOR mRNA after 2 - to - 24 h incubation with 100 μM H2O2 . Results shown are the means ± SEM of n = 3 experiments run in triplicate , analyzed using ANOVA with Dunnett multiple comparisons test . n . s . , not significant .

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D. RT-qPCR analyses of GSNOR mRNA after 2-to-24 h incubation with 100 μM H2O2. Results shown are the means ± SEM of n = 3 experiments run in triplicate, analyzed using ANOVA with Dunnett multiple comparisons test. n.s., not significant.


E . Western blot analysis of GSNOR in HEK293 cells treated for 8 h with 100 μM H2O2 in the presence or absence of trigonelline ( Trig ) . Densitometry of GSNOR immunoreactive bands is normalized to GADPH , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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E. Western blot analysis of GSNOR in HEK293 cells treated for 8 h with 100 μM H2O2 in the presence or absence of trigonelline (Trig). Densitometry of GSNOR immunoreactive bands is normalized to GADPH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01.


HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of cycloheximide ( CHX , F ) , GSNOR was evaluated by Western blot . Vertical dotted lines represent a virtual division of the nitrocellulose filter , as immunoreactive bands reported in figure - although part of the same experiment / gel - were not contiguous . Vehicle : PBS . Densitometry of GSNOR immunoreactive bands is normalized to Vinculin , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 4 independent experiments . * , p < 0 . 05 ; * * * , p < 0 . 001 ; n . s . , not significant .

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

HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of cycloheximide (CHX, F), GSNOR was evaluated by Western blot. Vertical dotted lines represent a virtual division of the nitrocellulose filter, as immunoreactive bands reported in figure - although part of the same experiment/gel - were not contiguous. Vehicle: PBS. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 4 independent experiments. *, p<0.05; ***, p<0.001; n.s., not significant.


G . HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of the proteasome inhibitor MG132 ( G ) . GSNOR was evaluated by Western blot . Vertical dotted lines represent a virtual division of the nitrocellulose filter , as immunoreactive bands reported in figure - although part of the same experiment / gel - were not contiguous . Vehicle : PBS . Densitometry of GSNOR immunoreactive bands is normalized to Vinculin , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 4 independent experiments . * , p < 0 . 05 ; * * * , p < 0 . 001 ; n . s . , not significant .

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

G. HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of the proteasome inhibitor MG132 (G). GSNOR was evaluated by Western blot. Vertical dotted lines represent a virtual division of the nitrocellulose filter, as immunoreactive bands reported in figure - although part of the same experiment/gel - were not contiguous. Vehicle: PBS. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 4 independent experiments. *, p<0.05; ***, p<0.001; n.s., not significant.


H . HEK293 cells were treated for 8 h ( blue ) or 24 h ( red ) with 100 μM H2O2 . Polysome profile showing monosomes and polysomes was obtained from control ( Vehicle ) and H2O2 treated lysates ( for either 8 or 24 h ) by separation on 5 - 50 % sucrose linear density gradient and collection using a gradient fractionation system .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 h (blue) or 24 h (red) with 100 μM H2O2. Polysome profile showing monosomes and polysomes was obtained from control (Vehicle) and H2O2 treated lysates (for either 8 or 24 h) by separation on 5-50% sucrose linear density gradient and collection using a gradient fractionation system.


I . RT - qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2 - treated samples ( for either 8 or 24 h ) . H3A mRNA expression level was used as housekeeping gene control . Results represent the means ± SD of n ≥ 6 independent experiments shown as % normalized by H3A . * * * , p < 0 . 001 Vehicle : PBS .

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

I. RT-qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2-treated samples (for either 8 or 24 h). H3A mRNA expression level was used as housekeeping gene control. Results represent the means ± SD of n ≥ 6 independent experiments shown as % normalized by H3A. ***, p<0.001 Vehicle: PBS.


J . HEK293 cells treated for 8 h or 24 h with 100 μM H2O2 in the presence or absence of puromycin ( tyrosyl - tRNA mimic used to label nascent proteins ) , or cycloheximide ( CHX , used to block protein translation ) . Nascent polypeptides were assessed by Western blot performed with an anti - puromycin ( anti - PMC ) antibody . Vehicle : PBS . β - actin was used as loading control .

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

J. HEK293 cells treated for 8 h or 24 h with 100 μM H2O2 in the presence or absence of puromycin (tyrosyl-tRNA mimic used to label nascent proteins), or cycloheximide (CHX, used to block protein translation). Nascent polypeptides were assessed by Western blot performed with an anti-puromycin (anti-PMC) antibody. Vehicle: PBS. β-actin was used as loading control.


A . HEK293 cells treated for 24 h with 100 μM H2O2 . Basal and phospho - active forms of ATM and CHK2 were assessed by Western blot . Phospho : basal level ratios of ATM and CHK2 immunoreactive bands are normalized to Vinculin , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n ≥ 4 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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 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 O O

A. HEK293 cells treated for 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2 were assessed by Western blot. Phospho:basal level ratios of ATM and CHK2 immunoreactive bands are normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n ≥ 4 independent experiments. *, p<0.05; **, p<0.01.


B . HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 ( KU ) . Basal and phospho - active forms of ATM , CHK2 , and GSNOR were assessed by Western blot . Vinculin and GAPDH were used as loading controls . Phospho : basal level ratios of ATM and CHK2 ( normalized to Vinculin ) , along with densitometry of GSNOR immunoreactive bands ( normalized to GAPDH ) are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 .

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

B. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 (KU). Basal and phospho-active forms of ATM, CHK2, and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Phospho:basal level ratios of ATM and CHK2 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.


C . HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the CHK2 inhibitor AZD7762 ( AZD ) . Basal and phospho - active forms of CHK2 , together with GSNOR were assesses by Western blot . Phospho : basal level ratios of CHK2 and densitometry of GSNOR immunoreactive bands are normalized to GAPDH , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

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

C. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the CHK2 inhibitor AZD7762 (AZD). Basal and phospho-active forms of CHK2, together with GSNOR were assesses by Western blot. Phospho:basal level ratios of CHK2 and densitometry of GSNOR immunoreactive bands are normalized to GAPDH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.


D . HEK293 cells were transfected with a pool of siRNA against ATM ( siATM ) or with control siRNA ( siScr ) for 18 h and treated for additional 24 h with 100 μM H2O2 . Basal and phospho - active forms of ATM , together with GSNOR were assessed by Western blot . Vinculin and GAPDH were used as loading controls . Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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 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

D. HEK293 cells were transfected with a pool of siRNA against ATM (siATM) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM, together with GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01.


E . HEK293 cells were transfected with a pool of siRNA against CHK2 ( siCHK2 ) or with control siRNA ( siScr ) for 18 h and treated for additional 24 h with 100 μM H2O2 . Basal and phospho - active forms of CHK2 , together with GSNOR were assessed by Western blot . GAPDH was used as loading control . Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 .

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 .

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

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 .

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

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 .

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

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.


[ A ] The same MVs from GB03 caused opposed impacts on plants grown in different medium . The petri dishes contain plastic partitions ( red dotted lines ) that separate different medium . 0 . 5 MS and 0 . 05 MS indicate 1 / 2 - strength ( nutrient - sufficient ) and 1 / 20th - strength ( nutrient - deficient ) Murashige & Skoog medium . Images were taken at 11 days after treatments ( DAT ) .

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[A] The same MVs from GB03 caused opposed impacts on plants grown in different medium. The petri dishes contain plastic partitions (red dotted lines) that separate different medium. 0.5 MS and 0.05 MS indicate 1/2-strength (nutrient-sufficient) and 1/20th-strength (nutrient-deficient) Murashige & Skoog medium. Images were taken at 11 days after treatments (DAT).


[ B ] Quantification of total leaf area per plant ( square centimeter , cm2 ) . Values correspond to the means ± SE of three biological replicates . Different letters denote significant differences at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

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[B] Quantification of total leaf area per plant (square centimeter, cm2). Values correspond to the means ± SE of three biological replicates. Different letters denote significant differences at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ C ] Effective quantum yield of photosystem II in plants . n = 3 biological replicates , mean ± SE . Asterisks denote significant differences at * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 , Dunnett ' s multiple comparison test ( comparing with the control ) within each group of the same DAT

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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] Effective quantum yield of photosystem II in plants. n=3 biological replicates, mean± SE. Asterisks denote significant differences at * p < 0.05, ** p < 0.01 and *** p < 0.001, Dunnett's multiple comparison test (comparing with the control) within each group of the same DAT


[ D ] Cell death visualization by trypan blue staining of 11 DAT leaves . Bar = 1mm

[D] Cell death visualization by trypan blue staining of 11 DAT leaves. Bar= 1mm


[ A ] Plants grown with 0 . 05 MS and GMVs ( G 0 . 05 ) showed hyper - induction of IPS1 and PS2 gene expression in both shoots and roots , compared to plants grown with 0 . 05 MS alone ( C 0 . 05 ) . Values are means ± SE of three biological replicates .

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

[A] Plants grown with 0.05 MS and GMVs (G 0.05) showed hyper-induction of IPS1 and PS2 gene expression in both shoots and roots, compared to plants grown with 0.05 MS alone (C 0.05). Values are means ± SE of three biological replicates.


[ B ] Root acid phosphatase activity as detected by the blue color from the 5 - bromo - 4 - chloro - 3 - indolyl phosphate p - toluidine salt ( BCIP ) treatment . Image contrast enhanced for improved visualization of the blue color in roots

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[B] Root acid phosphatase activity as detected by the blue color from the 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) treatment. Image contrast enhanced for improved visualization of the blue color in roots


Supplementation of Pi to the nutrient - deficient plants ( 0 . 05 + Pi ) significantly reduced hyper - accumulation of anthocyanin ( C ) triggered by GMVs . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

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

Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-accumulation of anthocyanin (C) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


Supplementation of Pi to the nutrient - deficient plants ( 0 . 05 + Pi ) significantly reduced hyper - induction of IPS1 gene expression ( D ) triggered by GMVs . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

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

Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-induction of IPS1 gene expression (D) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ E ] Pi supplementation to the 0 . 05 MS medium partially restored GMV - induced plant vigor , as indicated by increases in total leaf area per plant . [ F ] A low Pi level ( LP ) , which was equal to 1 / 20 of that in the 0 . 5 MS medium , in plant growth medium blocked GMV - induced plant vigor . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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] Pi supplementation to the 0.05 MS medium partially restored GMV-induced plant vigor, as indicated by increases in total leaf area per plant. [F] A low Pi level (LP), which was equal to 1/20 of that in the 0.5 MS medium, in plant growth medium blocked GMV-induced plant vigor. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ G ] The Arabidopsis phr1phl1 mutant showed substantially decreased growth - promotion , compared to the wild type plants . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

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

[G] The Arabidopsis phr1phl1 mutant showed substantially decreased growth-promotion, compared to the wild type plants. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ A ] Images of Arabidopsis plants exposed to four individual GMV components , including diacetyl ( DA ) , 2 , 3 - butanediol ( BTDL ) , 2 - methyl - 1 - propanol ( MP ) and acetoin ( ATN ) . The compounds were applied at dosages that , when the compounds totally evaporate from the agar - containing solid droplets , would yield in volatile concentrations of 9 . 7 µg ( DA ) , 32 . 5 µg ( BTDL ) , 7 . 9 µg ( MP ) and 28 . 5 µg ( ATN ) per mL free space in the petri dish , which resembled the 1 : 3 . 3 : 0 . 8 : 2 . 9 ratio among the four GMV components in natural GMVs22 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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] Images of Arabidopsis plants exposed to four individual GMV components, including diacetyl (DA), 2,3-butanediol (BTDL), 2-methyl-1-propanol (MP) and acetoin (ATN). The compounds were applied at dosages that, when the compounds totally evaporate from the agar-containing solid droplets, would yield in volatile concentrations of 9.7 µg (DA), 32.5 µg (BTDL), 7.9 µg (MP) and 28.5 µg (ATN) per mL free space in the petri dish, which resembled the 1 : 3.3 : 0.8 : 2.9 ratio among the four GMV components in natural GMVs22.


anthocyanin hyper accumulation ( B ) were observed in Pi - deficient plants exposed to DA but not to the other three GMV components . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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

anthocyanin hyper accumulation (B) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


PSR gene hyper induction ( C ) were observed in Pi - deficient plants exposed to DA but not to the other three GMV components . Data information : qPCR results show values of means ± SE of two biological replicates . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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

PSR gene hyper induction (C) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Compared to the wild type plants , phr1phl1 showed substantially decreased anthocyanin hyper accumulation ( D ) triggered by DA . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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

Compared to the wild type plants, phr1phl1 showed substantially decreased anthocyanin hyper accumulation (D) triggered by DA. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Compared to the wild type plants , phr1phl1 showed substantially decreased PSR gene hyper induction ( E ) triggered by DA . Data information : qPCR results show values of means ± SE of two biological replicates . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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

Compared to the wild type plants, phr1phl1 showed substantially decreased PSR gene hyper induction (E) triggered by DA. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


[ A ] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency . Data information : qPCR results show values of means ± SE ( n = 3 ) , two biological replicates were analyzed with similar results . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


[ B ] DA elevates SA accumulation levels in Pi - deficient Arabidopsis . Means ± SE of three biological replicates . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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[B] DA elevates SA accumulation levels in Pi-deficient Arabidopsis. Means ± SE of three biological replicates. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.