( F ) extent of colocalisation between GFP - Tollip LAMP1 from untreated AO treated SH - SY5Y were quantitated from immunofluorescence represented as a Pearson ' s correlation ( 2 - 3 per experiment , from 2 independent experiments ) . ( G - I ) extent of colocalisation between GFP - Tollip LAMP1 in Tom1 KO ( G ) , ATG5 KO ( H ) , VPS35 KO ( I ) compared to wild - type ( WT ) HeLa was quantified from immunofluorescence immunostained for GFP LAMP1 represented as a Pearson ' s correlation ( 2 - 3 per experiment , from 3 independent experiments ) . Western blot of lysates harvested from wild - type ( WT ) alongside either Tom1 KO ( G ) or VPS35 KO ( I ) Hela was performed using antibodies specific to indicated .

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

(F) The extent of colocalisation between GFP-Tollip and LAMP1 from untreated and AO treated SH-SY5Y cells were quantitated from immunofluorescence images and represented as a Pearson's correlation (2-3 cells per experiment, from 2 independent experiments). (G-I) The extent of colocalisation between GFP-Tollip and LAMP1 in Tom1 KO (G), ATG5 KO (H), and VPS35 KO (I) compared to wild-type (WT) HeLa cells was quantified from immunofluorescence images immunostained for GFP and LAMP1 and represented as a Pearson's correlation (2-3 cells per experiment, from 3 independent experiments). Western blot analysis of lysates harvested from wild-type (WT) alongside either Tom1 KO (G) or VPS35 KO (I) Hela cells was performed using antibodies specific to the indicated proteins.


( A ) A subset of TOM20 + ve / PDH - ve MDVs are trafficked to lysosome . siRNA knockdown of Tollip was conducted in SH - SY5Y over a 72 hours period prior to treatment with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or 25 μM antimycin A ( AA ) for 2 hours . were then fixed stained with antibodies specific to LAMP1 ( green ) , PDH E2 / E3bp ( red ) TOM20 ( blue ) . Arrows denote TOM20 + ve / PDH - ve MDVs colocalising with LAMP1 , whereas arrowheads denote MDVs that do not colocalise with LAMP1 . ( B ) From immunofluorescence , total number of TOM20 + ve / PDH - ve MDVs were counted as well as number of TOM20 MDVs that colocalised with LAMP1 to calculate % of TOM20 positive MDVs colocalising with LAMP1 per ( n = 25 - 32 per condition from 3 independent experiments ) .

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

(A) A subset of TOM20+ve/PDH-ve MDVs are trafficked to the lysosome. siRNA knockdown of Tollip was conducted in SH-SY5Y cells over a 72 hours period prior to treatment with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM antimycin A (AA) for 2 hours. Cells were then fixed and stained with antibodies specific to LAMP1 (green), PDH E2/E3bp (red) and TOM20 (blue). Arrows denote TOM20+ve/PDH-ve MDVs colocalising with LAMP1, whereas arrowheads denote MDVs that do not colocalise with LAMP1. (B) From immunofluorescence images, the total number of TOM20+ve/PDH-ve MDVs were counted as well as the number of TOM20 MDVs that colocalised with LAMP1 to calculate the % of TOM20 positive MDVs colocalising with LAMP1 per cell (n = 25-32 cells per condition from 3 independent experiments).


( C ) siRNA knockdown was performed in SH - SY5Y , then 18 hours prior to fixation were transfected with GFP - Tollip wild - type ( WT ) , ∆ Nterm , CUEmut , or R78A . were fixed immunostained for PDH E2 / E3bp ( red ) TOM20 ( blue ) . Arrowheads denote TOM20 + ve / PDH - ve MDVs . ( D ) of TOM20 MDVs per was performed by eye ( n = 11 - 14 per mutant from 2 independent experiments ) . expressing a GFP - Tollip construct were identified using GFP channel . Surrounding not expressing GFP were counted as Tollip siRNA only population .

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

(C) siRNA knockdown was performed in SH-SY5Y cells, then 18 hours prior to fixation cells were transfected with GFP-Tollip wild-type (WT), ∆Nterm, CUEmut, or R78A. Cells were fixed and immunostained for PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs. (D) Quantification of TOM20 MDVs per cell was performed by eye (n = 11-14 cells per mutant from 2 independent experiments). Cells expressing a GFP-Tollip construct were identified using the GFP channel. Surrounding cells not expressing GFP were counted as the Tollip siRNA only population.


( A ) GSCs contain higher of stemness markers ( Nestin , SOX2 , Musashi - 1 , NANOG , OCT4 ) compared with their DFCs . Total RNAs from GSCs DFCs were subjected to real - time PCR . average relative amounts of GSCs were normalized with those of DFCs . Shown are means + S . D . of normalized mRNA of GSCs obtained from three independent experiments .

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

(A) GSCs contain the higher levels of stemness markers (Nestin, SOX2, Musashi-1, NANOG, and OCT4) compared with their DFCs. Total RNAs from GSCs and DFCs were subjected to real-time PCR. The average relative amounts of GSCs were normalized with those of DFCs. Shown are means +S.D. of the normalized mRNA levels of GSCs obtained from the three independent experiments.


( B ) Immunoblotting of markers for stemness ( SOX2 , Nestin Musashi - 1 ) differentiation ( GFAP TUJ1 ( Neuron - specific class III beta - tubulin ) ) in GSCs in comparison with GSC - derived at different passage numbers ( P1 - P7 ) .

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

(B) Immunoblotting analysis of markers for stemness (SOX2, Nestin and Musashi-1) and differentiation (GFAP and TUJ1 (Neuron-specific class III beta-tubulin)) in GSCs in comparison with GSC-derived cells at different passage numbers (P1-P7).


( C ) Measurement of doubling time . Approximately 3 × 104 for each of XO8 GSCs XO8 DFCs were seeded in a 60 mm culture dish counted every day . were treated with trypsin - EDTA , number of viable was counted with a hemocytometer . doubling time was calculated from growth curve over 5 days . Shown are means ± S . D . obtained from three independent experiments .

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(C) Measurement of the cell doubling time. Approximately 3×104 cells for each of XO8 GSCs and XO8 DFCs were seeded in a 60 mm culture dish and counted every day. The cells were treated with trypsin-EDTA, and the number of viable cells was counted with a hemocytometer. The doubling time was calculated from the cell growth curve over 5 days. Shown are means ±S.D. obtained from three independent experiments.


( D ) FACS of CD133 GFAP using X08 GSCs DFCs . Data are from representative experiments repeated at least three times .

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(D) FACS analysis of CD133 and GFAP using X08 GSCs and DFCs. Data are from representative experiments repeated at least three times.


( E ) Immunoblotting of CD133 GFAP in GSCs in comparison with DFCs .

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(E) Immunoblotting analysis of CD133 and GFAP in GSCs in comparison with DFCs.


( F ) Quantitation of ( G ) . ( G ) Equal numbers of XO8 GSCs DFCs were injected orthotropically into mouse brains . After six , brains were harvested tumorigenic potential was assessed by evaluating formation . Representative microphotographs of brain sections of mice injected with XO8 GSCs XO8 DFC controls .

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(F) Quantitation of (G). (G) Equal numbers of XO8 GSCs and DFCs were injected orthotropically into mouse brains. After six weeks, brains were harvested and tumorigenic potential was assessed by evaluating tumor formation. Representative microphotographs of the brain sections of mice injected with XO8 GSCs and XO8 DFC controls.


viability assays were performed as described in Materials Methods after were treated with temozolomide ( A ) , cisplatin ( B ) , adriamycin ( C ) , taxol ( D ) , TRAIL ( E ) , bafilomycin A1 ( F ) , thapsigargin ( G ) , MG132 ( H ) . Means ± S . D . from at least three independent experiments are shown as relative indexes after normalization to those of control treated with DMSO .

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Cell viability assays were performed as described in Materials and Methods after cells were treated with temozolomide (A), cisplatin (B), adriamycin (C), taxol (D), TRAIL (E), bafilomycin A1 (F), thapsigargin (G), and MG132 (H). Means ±S.D. from at least three independent experiments are shown as relative indexes after normalization to those of individual control cells treated with DMSO.


( I ) XO8 GSCs DFCs were treated with 50 nM MG132 for 24 hrs . viability was measured using trypan blue exclusion . Error bars represent mean ± SD from three independent experiments . For statistical , student t - ( two - sided , one type ) were used . ( * P < 0 . 05 ) .

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(I) XO8 GSCs and DFCs were treated with 50 nM MG132 for 24 hrs. Cell viability was measured using trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. (*P < 0.05).


( J ) XO8 GSCs were treated with MG132 were cultured in 0 . 3 % soft - agar as described in Materials Methods . colonies bigger than 100 µm in diameter in four randomly chosen fields were subjected to blind counting . Bars , means + S . D . from three independent experiments .

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(J) XO8 GSCs were treated with MG132 were cultured in 0.3% soft-agar as described in Materials and Methods. The colonies bigger than 100 µm in diameter in four randomly chosen fields were subjected to blind counting. Bars, means +S.D. from three independent experiments.


( A ) XO8 GSCs DFCs were differentially labeled with PKH67 ( green ) PKH26 ( red ) , respectively , mixed before plated on laminine - coated plates . were treated with 50 nM MG132 subjected to microscopy . Scale bar , 100 μM . ( B ) Quantitation of ( A ) . numbers from three independent experiments were normalized to those of control treated with DMSO presented as means + S . D . ( * p = 0 . 0035 , n = 3 ) . Student t - ( two - sided , one type ) were used for statistical .

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

(A) XO8 GSCs and DFCs were differentially labeled with PKH67 (green) and PKH26 (red), respectively, and mixed before plated on laminine-coated plates. The cells were treated with 50 nM MG132 and subjected to fluorescent microscopy. Scale bar, 100 μM. (B) Quantitation of (A). Cell numbers from three independent experiments were normalized to those of control cells treated with DMSO and presented as means +S.D. (*p=0.0035, n=3). Student t-test (two-sided, one type) were used for statistical analysis.


( C ) harvested from ( A ) were subjected to FACS .

(C) The harvested cells from (A) were subjected to FACS analysis.


( D ) XO8 GSCs were treated with various PIs ( salinosporamide A , epoxomicin , PS341 ) , followed by viability assays . Bars , means ± S . D . from three independent experiments .

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

(D) XO8 GSCs were treated with various PIs (salinosporamide A, epoxomicin, and PS341), followed by cell viability assays. Bars, means ±S.D. from three independent experiments.


( E ) XO6 , XO8 , XO10 GSCs were treated with MG132 subjected to viability . Bars , means ± S . D . from three independent experiments . ( F ) XO6 , XO8 , XO10 GSCs were treated with MG132 subjected to viability in serum - containing media . Bars , means ± S . D . from three independent experiments .

O O O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(E) XO6, XO8, and XO10 GSCs were treated with MG132 and subjected to cell viability assay. Bars, means ±S.D. from three independent experiments. (F) XO6, XO8, and XO10 GSCs were treated with MG132 and subjected to cell viability assay in serum-containing media. Bars, means ±S.D. from three independent experiments.


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(A) Immunoblotting of XO8 GSCs and DFCs.


( B ) Immunoblotting of Ub in XO8 GSCs XO8 GSC - derived in course of differentiation ( P1 - P7 ) . ( C ) Similar to ( B ) except that XO6 GSCs were used .

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(B) Immunoblotting analysis of Ub in XO8 GSCs and XO8 GSC-derived cells in the course of differentiation (P1-P7). (C) Similar to (B) except that XO6 GSCs were used.


( D ) Immunoblotting of ubiquitin ligases proteasomal subunits in XO8 GSCs XO8 GSC - derived in course of differentiation ( P1 - P7 ) . ( E ) Similar to ( E ) except that XO6 GSCs were used .

O O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O

(D) Immunoblotting analysis of ubiquitin ligases and proteasomal subunits in XO8 GSCs and XO8 GSC-derived cells in the course of differentiation (P1-P7). (E) Similar to (E) except that XO6 GSCs were used.


( F - I ) In vitro proteasome activity assays were performed as described in Materials Methods . Bars represent means + S . D . from three independent experiments . For statistical , student t - ( two - sided , one type ) were used . * P value : ( F ) , 0 . 0039 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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.


( F - I ) In vitro proteasome activity assays were performed as described in Materials Methods . Bars represent means + S . D . from three independent experiments . For statistical , student t - ( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-CELL_LINE O O O O O O O O O B-CELL_LINE O O O O O

(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 XO8 DFCs treated with MG132 as described in Materials Methods were labeled with annexin V visualized using 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 ) from ( J ) were subjected to flow cytometry . Annexin V - positive numbers were obtained from three independent experiments normalized to those of controls treated with DMSO . Bars , means + S . D . from three independent experiments . For statistical , student t - ( two - sided , one type ) were used . * P value : 6 hrs , 0 . 0012 ; 17 hrs , 0 . 0041 .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O

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


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

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


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

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

(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 of proteasomal subunits in XO8 GSCs in comparison with XO8 DFCs at passages 11 12 . Note that part of Figure 5C is also shown as left panel of Figure 4A . ( D ) Same as ( C ) except that 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 of NF - κB pathway in XO8 GSCs XO8 DFCs .

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


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


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(G) Immunoblotting analysis of JNK and p38.


( H ) viability was performed as in Fig . 2 with 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 treated with DMSO . For statistical , student t - ( two - sided , one type ) were used . * * P value = 0 . 724 , 0 . 502 , 0 . 439 for 0 . 5 , 1 . 0 5 . 0 μM SB203580 , respectively at 0 . 1 μM MG132 , 0 . 760 , 0 . 683 , 0 . 498 for 0 . 5 , 1 . 0 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 treated with DMSO . For statistical , student t - ( two - sided , one type ) were used . * P value = 0 . 009 , 0 . 002 , 0 . 07 for 0 . 5 , 1 . 0 5 . 0 μM SP600129 , respectively at 0 . 1 μM MG132 , 0 . 018 , 0 . 025 , 0 . 09 for 0 . 5 , 1 . 0 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 of XO8 GSCs treated with 50 nM MG132 alone or in combination with 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 B ) XO8 GSCs XO8 DFCs were treated with 50 nM MG132 for indicated hours . expression of genes was assessed by real - time PCR using total extracted from MG132 - treated . Mean + S . D . values from three independent experiments are shown after normalization to that of control 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 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 . lysates were subjected to immunoblot with anti - PARP - 1 , anti - CHOP , anti - ATF3 , anti - ATF3 , 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 were treated with 50 nM MG132 in absence or presence of 5μM actinomycin D ( ActD ) for 6 h . 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 were transfected with ATF3 shRNA or vector ( pLKO1 ) , treated with 50 nM MG132 , 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 Methods . After four days , individualized GSCs were treated with 50 nM MG132 , 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 then treated with 50 nM MG132 for 24 hrs . viability was determined using Trypan blue exclusion . Error bars represent mean ± SD from three independent experiments ( * P < 0 . 05 , n = 3 ) . For statistical , student t - ( 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 of ( C ) . were collected , lysed , subjected to immunoblot 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 CHOP - / - MEF were treated with MG132 for 24 hrs . viability was determined using trypan blue exclusion . Error bars represent mean ± SD from three separate experiments ( * P < 0 . 05 , n = 3 ) . For statistical , student t - ( 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 ) lysates obtained in ( E ) were subjected to immunoblot 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 ) expression of NOXA was assessed with real - time PCR using total 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 ) . mean + S . D . values from three independent experiments are shown after normalization to that of control treated with DMSO . For statistical , student t - ( two - sided , one type ) was used . * P value : salubrinal , 0 . 0003 ; STK064650 , 0 . 0036 ; STK047915 , 0 . 0019 .

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

(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 . mean + S . D . values from three independent experiments are shown after normalization to that of control treated with DMSO . For statistical , student t - ( 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 ) , 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 B ) XO8 GSCs were subcutaneously ( s . c . ) injected in flank of mice . When volume reached approximately 180 mm3 , GSC - bearing mice were administered intraperitoneally ( i . p . ) with 1 mg / kg MG132 10 mg / kg STK alone or in combination on every day for 2 . Tumors were dissected from subcutaneous regions of nude mice . Inset picture shows in respective group . Therapeutic effects were monitored using bioluminescence imaging . ( C ) bar diagram represents volumes . ( D ) weights were determined at end of study . ( E ) sizes in each group were determined by histological at day 21 after 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 brains of BALB / c nude mice . STK047915 , MG132 , or both were injected intraperitoneally to GSCs - bearing mice at day 14 after XO10 ( 5 × 105 ) inoculation . PBS was used as a control . Representative of H & amp ; amp ; E from each group show 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 were treated for 8 24 h with 100 μM H2O2 . GSNOR was evaluated by Western blot . Densitometry of each lane is normalized to GADPH , selected as loading control , expressed as arbitrary units . Values shown represent means ± SD of n ≥ 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 calculated with regard to H2O2 - untreated .

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

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

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

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

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 means ± of n = 3 experiments run in triplicate , analyzed using ANOVA with Dunnett multiple comparisons . 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 of GSNOR in HEK293 treated for 8 h with 100 μM H2O2 in presence or absence of trigonelline ( Trig ) . Densitometry of GSNOR immunoreactive bands is normalized to GADPH , selected as loading control , expressed as arbitrary units . Values shown represent means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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

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

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

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

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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 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 means ± SD of n ≥ 6 independent experiments shown as % normalized by H3A . * * * , p < 0 . 001 Vehicle : PBS .

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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 treated for 8 h or 24 h with 100 μM H2O2 in presence or absence of puromycin ( tyrosyl - tRNA mimic used to label nascent ) , or cycloheximide ( CHX , used to block 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 B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD 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 treated for 24 h with 100 μM H2O2 . Basal phospho - active forms of ATM CHK2 were assessed by Western blot . Phospho : basal level ratios of ATM CHK2 immunoreactive bands are normalized to Vinculin , selected as loading control , expressed as arbitrary units . Values shown represent means ± SD of n ≥ 4 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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

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

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

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

O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were transfected with a pool of siRNA against CHK2 ( siCHK2 ) or with control siRNA ( siScr ) for 18 h treated for additional 24 h with 100 μM H2O2 . Basal 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 expressed as arbitrary units . Values shown represent means ± SD of n = 3 independent experiments . * , p < 0 . 05 .

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

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 were treated for 24 h with 100 μM H2O2 in presence or absence of ATM inhibitor KU55933 ( KU ) . Lysates were subjected to biotin - switch , S - nitrosylated ( PSNOs ) revealed by incubation with horseradish peroxidase ( HRP ) - conjugated streptavidin . Results obtained in absence of ascorbate ( - ASC ) are shown as negative controls . Densitometry of each lane is normalized to LDH , selected as loading control , expressed as arbitrary units . Values shown represent means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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

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 heavy polysome fraction pooled together in H2O2 treated samples in absence or in presence of ATM inhibitor KU55933 ( KU ) . H3A mRNA expression level was used as housekeeping gene control . Results are means ± of n = 3 independent experiments run in duplicate shown as % , normalized by H3A . * , p < 0 . 05 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 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 , expressed as arbitrary units ( with HU arbitrarily set to 1 ) . Values shown represent means ± SD of n = 3 independent experiments . n . s . , not significant .

O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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 were transfected with a pool of siRNA against CHK1 ( siCHK1 ) or with control siRNA ( siScr ) for 18 h treated for additional 24 h with 100 μM H2O2 . CHK1 GSNOR were assessed by Western blot of . Densitometry of GSNOR immunoreactive bands is normalized to Vinculin , selected as loading control , expressed as arbitrary units . Values shown represent means ± SD of n = 3 independent experiments . * , p < 0 . 05 .

O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 treated for 4 h with 100 μM H2O2 . Phospho - histone H2A . X ( γH2A . X ) was assessed by immunofluorescence . Nuclei ( blue ) were stained with Hoechst 33342 . scale bar : 10μM . 4X digital magnification is shown at bottom left of each image .

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

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 indicated time points . GAPDH was used as loading control ( top panel ) . Densitometry of each lane is normalized to GAPDH , selected as loading control , expressed as % of γH2A . X in H2O2 - treated versus untreated . Values represent means ± SD of two independent experiments ( bottom panel ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE 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 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 phospho - active forms of ATR CHK1 ( at Ser345 ) were assessed by Western blot . GAPDH was used as loading control .

O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD 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 were treated for 8 h with 20 μM neocarzinostatin ( NCS ) . ( left ) Western blot of basal phospho - active forms of ATM , CHK2 , ATR , CHK1 , p53 , GSNOR in HEK293 treated for 8 h with 20 μM neocarzinostatin ( NCS ) . Vinculin 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 means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

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

D. 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 were treated for 8 h with 20 μM

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


E . HEK293 were treated for 2 , 4 8 h with 20 μM neocarzinostatin ( NCS ) . After treatment , were incubated with 2 ' , 7 ' - H2DCF - DA to cytofluorometrically assess intracellular production of H2O2 . Values are shown as units of DCF relative to untreated . Values are shown as units of relative to NCS - untreated ( arbitrarily set as 1 ) represent means ± SD of n = 3 independent experiments . n . s . , not significant .

O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 were transfected with plasmids coding for wild - type ( WT ) , C2991L redox mutant ( CL ) of ATM , or with an empty vector ( Empty ) ( used as negative control ) for 40 h treated for additional 8 h with 100 μM H2O2 . Basal phospho - active forms of ATM , GSNOR were assessed by Western blot . Vinculin GAPDH were used as loading controls . Two different exposures ( low high ) were selected to highlight differences in P - ATM in different experimental settings .

O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 were treated for 24 h with 100 μM H2O2 in presence or absence of 20 μM pifithrin - α ( Pft ) . Basal phospho - active forms of ATM , CHK2 , p53 , GSNOR were assessed by Western blot . Phospho : basal level ratios of ATM , CHK2 p53 ( normalized to Vinculin ) , along with densitometry of GSNOR immunoreactive bands ( normalized to GAPDH ) are expressed as arbitrary units . Values shown represent means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were transfected with a pool of siRNA against p53 ( sip53 ) or with control siRNA ( siScr ) for 24 h treated for additional 24 h with 100 μM H2O2 . Basal phospho - active forms of ATM CHK2 , as well as p53 GSNOR were assessed by Western blot . Phospho : basal level ratios of ATM CHK2 ( normalized to Vinculin ) , along with densitometry of GSNOR immunoreactive bands ( normalized to GAPDH ) are expressed as arbitrary units . Values shown represent means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expressing 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 phospho - active forms of ATM CHK2 , p53 GSNOR were assessed by Western blot . Vinculin GAPDH were used as loading controls . Phospho : basal level ratios of ATM CHK2 , along with densitometry of p53 GSNOR immunoreactive bands are expressed as arbitrary units . Values shown represent means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 treated for 2 , 4 8 h with 100 μM H2O2 . After treatment , were incubated with 5 μM 2 ' , 7 ' - H2DCF - DA ( A ) or MitoSox ( B ) to evaluate production of H2O2 or mitochondrial superoxide , respectively . Values are shown as units of DCF or MitoSox relative to untreated ( arbitrarily set as 1 ) represent means ± SD of n ≥ 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

O O O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 mitophagy was assessed by Western blot of different mitochondrial subunits [ i . e . , NDUFB8 ( I ) , SDHB ( II ) , MTCO2 ( IV ) ] . Basal phospho ATM , as well as GSNOR were used to check efficiency of siRNA - mediated knock down . Vinculin was used as loading control .

O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O O O B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O 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 immunoreactive bands of panel C ( normalized to Vinculin ) is indicated as H2O2 - treated versus untreated ( CTR ) expressed as arbitrary units . Values shown represent means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . , not significant .

O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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 were depleted of endogenous ATM by repeated transfections with shRNA induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , were further transfected with a GSNOR - coding vector 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 means ± SD of n = 6 experiments . * p < 0 . 05 ; * * * p < 0 . 001 .

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

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 same experimental settings , mitophagy was also assessed at 8 h by microscopy analyses upon incubation with chloroquine ( CLQ ) to enhance differences in mitophagy . of mitochondria merging with LC3 - positive calculated by Fiji software using open - source plugin ComDet v . 0 . 3 . 7 . Values are expressed as % of mitochondria ( TOM20 + particles ) co - localizing with LC3 / graphed as boxes ( 25th - 75th interquartile range ) whiskers ( minimum to maximum showing points ) , with central bands representing median of n ≥ 7 different analyzed . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

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 same experimental settings , mitophagy was also assessed at 8 h by 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 B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-SUBCELLULAR 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 were treated with 10 μM CCCP for 8 h . After treatment , were incubated with 5 μM 2 ' , 7 ' - H2DCF - DA ( A ) or MitoSox ( B ) to evaluate production of H2O2 or mitochondrial superoxide , respectively . Values are shown as units of DCF or MitoSox relative to untreated ( arbitrarily set as 1 ) represent means ± SD of n = 3 independent experiments . * * * p < 0 . 001 .

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

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 same experimental settings , basal phospho - active forms of ATM , GSNOR were assessed by Western blot . Vinculin GAPDH were used as loading controls .

O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD 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 were depleted of endogenous ATM by repeated transfections with shRNA induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , were further transfected with a GSNOR - coding vector 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 means ± SD of n = 4 experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

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 were depleted of endogenous ATM by repeated transfections with shRNA induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , were further transfected with a GSNOR - coding vector then treated for 2 h with 10 μM CCCP . ( E 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 .

B-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-SUBCELLULAR 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 were depleted of endogenous ATM by repeated transfections with shRNA induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , were further transfected with a GSNOR - coding vector then treated for 2 h with 10 μM CCCP . F ) microscopy analyses upon incubation with chloroquine ( CLQ ) to enhance differences in mitophagy . of mitochondria merging with LC3 - positive calculated by Fiji software using open - source plugin ComDet v . 0 . 3 . 7 . Values are expressed as % of mitochondria ( TOM20 + particles ) co - localizing with LC3 / graphed as boxes ( 25th - 75th interquartile range ) whiskers ( minimum to maximum showing points ) , with central bands representing median of n ≥ 9 different . * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . , not significant .

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

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 were depleted of endogenous ATM by repeated transfections with shRNA induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , were further transfected with a GSNOR - coding vector then treated for 2 h with 10 μM CCCP . ( G Western blot of different subunits of mitochondrial , i . e . , NDUFB8 ( I ) , SDHA SDHB ( II ) , MTCO2 ( IV ) voltage - dependent anion channel ( VDAC ) . Tubulin , LDH Vinculin were used as loading controls .

B-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD 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 were depleted of endogenous ATM by repeated transfections with shRNA induced , by doxycycline incubation , to express ATMWT , ATM2RA , or ATMCL mutant . Where indicated , were further transfected with a GSNOR - coding vector then treated for 2 h with 10 μM CCCP . H ) Densitometry of mitochondrial immunoreactive bands expressed as arbitrary units . Values shown represent means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . , not significant .

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

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 overexpressing wild type form of GSNOR ( GSNORwt ) or an empty vector ( Empty ) were subjected to combined treatment ( 200 μM H2O2 + 400 μM DPTA ) in presence or absence of ATM inhibitor KU55933 ( KU ) . of dead were performed with Trypan blue exclusion . Western blot of GSNOR is shown as inset in graph to substantiate transfection efficiency . Data , shown as fold change of dead relative to untreated ( arbitrarily set to 1 ) , represent mean ± of n = 4 experiments done in duplicate * p < 0 . 05 ; with respect to Empty .

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

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 Empty upon 24 h treatment with H2O2 + DPTA in presence KU .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 of Caspase3 PARP1 in GSNORwt Empty treated as in panel C . Tubulin was used as loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD 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 overexpressing 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 ) viability assessed by Alamar blue ( AB ) . Data , shown as fold change of viable , refer to AB ( relative to untreated , arbitrarily set to 1 ) represent means ± SD of n = 6 independent experiments . * * , p < 0 . 01 ; n . s . , not significant .

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

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 ) Gsnor - null ( KO ) mice treated with 200 or 400 μM H2O2 for 24 h .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE 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 Gsnor KO MAFs were subjected to treatment with 200 μM H2O2 , or 400 μM DPTA , or a combination of both . viability was evaluated by LIVE / DEAD .

O B-GENEPROD O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY 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 Gsnor KO MAFs were subjected to treatment with 200 μM H2O2 , or 400 μM DPTA , or a combination of both . viability was evaluated by LIVE / DEAD . Scale bar = 50 µm . Data , shown as % of dead ( red ) , represent mean ± 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 B-GENEPROD O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE 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 were treated for 10 30 min with PMA ( 200 ng / ml ) ionomycin ( iono ) ( 300 ng / ml ) . After treatment , were incubated with 5 μM 2 ' , 7 ' - H2DCF - DA ( A ) or DAF - FM - DA ( B ) to evaluate production of H2O2 or NO , respectively . Values are expressed as units of DCF or DAF - FM relative to untreated ( arbitrarily set as 1 ) graphed as boxes ( 25th - 75th interquartile range ) whiskers ( minimum to maximum showing points ) , with central bands representing median of n = 4 ( A ) n = 6 ( B ) independent experiments . * p < 0 . 01 ; * * p < 0 . 001 ; * * * p < 0 . 001 .

O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 were treated for 24 h with H2O2 ( 50 μM ) or PMA / ionomycin ( 200 + 300 ng / ml ) . Basal phospho - active form of ATM , 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 means ± SD of n = 3 independent experiments . * , p < 0 . 05 .

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

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 were treated for 24 h with H2O2 ( 50 μM ) or PMA / ionomycin ( 200 + 300 ng / ml ) , in presence or absence of ATM inhibitor ( KU55933 ) ( 5 μM ) or GSNOR inhibitor ( N6022 ) ( 25 μM ) . was assessed cytofluorimetrically upon with propidium iodide ( PI ) . Values are expressed as % of sub - G1 population of PI - stained graphed as boxes ( 25th - 75th interquartile range ) whiskers ( minimum to maximum showing points ) , with central bands representing median of n = 5 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 .

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

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.