( F ) Images captured using super - resolution radial fluctuations ( SRRF ) microscopy showing both TOM20 ( arrowheads ) and PDH ( arrows ) MDVs in SH - SY5Y cells after Tollip siRNA knockdown . Left side images are the first in each of the original widefield image stacks . Scale bar for zoom images is 0 . 5 μm .

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

(F) Images captured using super-resolution radial fluctuations (SRRF) microscopy showing both TOM20 (arrowheads) and PDH (arrows) MDVs in SH-SY5Y cells after Tollip siRNA knockdown. Left side images are the first in each of the original widefield image stacks. Scale bar for zoom images is 0.5 μm.


( A ) TOM20 - positive MDV formation was induced in SH - SY5Y by siRNA knockdown of Tollip . MDVs were defined as TOM20 + ve / Cytochrome c ( Cyt c ) - ve via immunofluorescence microscopy , which indicated some colocalisation with Parkin ( arrowheads ) .

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(A) TOM20-positive MDV formation was induced in SH-SY5Y by siRNA knockdown of Tollip. MDVs were defined as TOM20+ve/Cytochrome c (Cyt c)-ve via immunofluorescence microscopy, which indicated some colocalisation with Parkin (arrowheads).


( B ) SH - SY5Y cells were transfected with GFP - Tollip for 24 hours , then treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or AO / 100 μM bafilomycin A1 ( BfnA1 ) for 2 hours prior to fixation and immunostaining . Antibodies specific to GFP ( green ) , Parkin ( red ) and ubiquitin ( blue ) were used .

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(B) SH-SY5Y cells were transfected with GFP-Tollip for 24 hours, then treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A1 (BfnA1) for 2 hours prior to fixation and immunostaining. Antibodies specific to GFP (green), Parkin (red) and ubiquitin (blue) were used.


C ) GFP - Tollip / Parkin colocalisation was quantified from cells under steady state conditions or treated with AO and AO / BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell , across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ) . Results are represented as a percentage of Tollip puncta positive for Parkin .

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C) GFP-Tollip/Parkin colocalisation was quantified from cells under steady state conditions or treated with AO and AO/BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell, across 3-6 GFP-transfected cells/condition, per experiment (n = 3). Results are represented as a percentage of Tollip puncta positive for Parkin.


( D ) SH - SY5Y cells expressing GFP - Tollip were immunostained for GFP ( green ) , Parkin ( red ) , and Rab7a ( blue ) . Arrowheads indicate areas of colocalisation between all three markers .

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(D) SH-SY5Y cells expressing GFP-Tollip were immunostained for GFP (green), Parkin (red), and Rab7a (blue). Arrowheads indicate areas of colocalisation between all three markers.


( E - F ) SH - SY5Y cells were transfected with GFP - Tollip R78A or CUE mutant for 24 hours , then treated with AO or AO / BfnA1 for 2 hours prior to fixation and

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(E-F) SH-SY5Y cells were transfected with GFP-Tollip R78A or CUE mutant for 24 hours, then treated with AO or AO/BfnA1 for 2 hours prior to fixation and immunostaining


( G ) The percentage of GFP - Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0162 , * * p = 0 . 0034 , * * * * p < 0 . 0001 ) .

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(G) The percentage of GFP-Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/ condition, per experiment (n = 3; *p=0.0162, **p=0.0034, ****p<0.0001).


( H ) The percentage of ubiquitin puncta positive for Tollip was quantitated from immunuofluorescence images immunostained for ubiquitin and GFP from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0382 , * * p = 0 . 0068 ) .

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(H) The percentage of ubiquitin puncta positive for Tollip was quantitated from immunuofluorescence images immunostained for ubiquitin and GFP from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0382, **p=0.0068).


( I ) The percentage of ubiquitin puncta positive for Parkin was quantitated from immunuofluorescence images immunostained for ubiquitin and Parkin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0232 , * * p = 0 . 0062 ) .

(I) The percentage of ubiquitin puncta positive for Parkin was quantitated from immunuofluorescence images immunostained for ubiquitin and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0232, **p=0.0062).


( J ) The percentage of GFP - Tollip / Parkin vesicles positive for ubiquitin were quantitated from immunofluorescence images immunostained for GFP , Parkin , and ubiquitin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * * p = 0 . 0070 ) .

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(J) The percentage of GFP-Tollip/Parkin vesicles positive for ubiquitin were quantitated from immunofluorescence images immunostained for GFP, Parkin, and ubiquitin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; **p=0.0070).


( A ) HeLa cells expressing mycBioID - Tollip and HA - Parkin were either left untreated or treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or AO / 100 μM bafilomycin A ( BfnA1 ) for 6 hours in the presence of biotin . Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins . Proteins in whole cell extracts and pulldowns from each condition were then separated by SDS - PAGE and membranes probed with specific antibodies . Immunoblotting for Tom1 and mycBioID - Tollip were used as positive controls . Cells cultured in media lacking biotin were used as a negative control to assess background levels . Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions .

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(A) HeLa cells expressing mycBioID-Tollip and HA-Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 hours in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole cell extracts and pulldowns from each condition were then separated by SDS-PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID-Tollip were used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions.


( B ) HeLa cells expressing mycBioID alone and HA - Parkin were used to confirm the specificity of the Tollip - Parkin interaction .

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(B) HeLa cells expressing mycBioID alone and HA-Parkin were used to confirm the specificity of the Tollip-Parkin interaction.


( C ) Hela cells co - expressing GFP - Tollip and HA - Parkin were either left untreated or treated with AO for 2 hours prior to lysis . Cell lysates were subjected to either a normal rabbit IgG IP or HA IP and western blot analysis was performed using antibodies against Parkin and GFP .

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(C) Hela cells co-expressing GFP-Tollip and HA-Parkin were either left untreated or treated with AO for 2 hours prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP and western blot analysis was performed using antibodies against Parkin and GFP.


( D ) HeLa cells expressing mycBioID - Tollip containing the CUE domain mutation ( CUEmut ) and HA - Parkin were subjected to biotinylation and streptavidin pulldowns , followed by western blot analysis .

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(D) HeLa cells expressing mycBioID-Tollip containing the CUE domain mutation (CUEmut) and HA-Parkin were subjected to biotinylation and streptavidin pulldowns, followed by western blot analysis.


( E ) HeLa wild - type ( WT ) , Tom1 KO and ATG5 KO cells expressing mycBioID - Tollip and HA - Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins .

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(E) HeLa wild-type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID-Tollip and HA-Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins.


( A ) Cells were reverse transfected with Parkin or Tom1 siRNA oligos for 24 hours before a second transfection was performed for a further 48 hours , then fixed and stained with antibodies specific to TOM20 ( green ) and PDH E2 / E3bp ( red ) . Nuclei are labelled with Hoechst ( blue ) . Zoom insets represent 5x magnification and arrowheads indicate TOM20 - positive MDVs .

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(A) Cells were reverse transfected with Parkin or Tom1 siRNA oligos for 24 hours before a second transfection was performed for a further 48 hours, then fixed and stained with antibodies specific to TOM20 (green) and PDH E2/E3bp (red). Nuclei are labelled with Hoechst (blue). Zoom insets represent 5x magnification and arrowheads indicate TOM20-positive MDVs.


( B ) Knockdowns were confirmed for both Parkin and Tom1 by western blotting .

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(B) Knockdowns were confirmed for both Parkin and Tom1 by western blotting.


( C , D ) Quantification of MDVs , defined as TOM20 + ve / PDH - ve , ( C ) or PDH + ve / TOM20 - ve ( D ) per cell under steady - state conditions were performed ( n = 32 - 52 cells per condition from 3 independent experiments ) .

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(C, D) Quantification of MDVs, defined as TOM20+ve/PDH-ve, (C) or PDH+ve/TOM20-ve (D) per cell under steady-state conditions were performed (n = 32-52 cells per condition from 3 independent experiments).


( E ) Western blot analysis of lysates harvested from SH - SY5Y wild - type ( empty vector ) and CRISPR generated Parkin KO cells . Immunoblot analysis was performed using antibodies against Parkin and GAPDH .

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(E) Western blot analysis of lysates harvested from SH-SY5Y wild-type (empty vector) and CRISPR generated Parkin KO cells. Immunoblot analysis was performed using antibodies against Parkin and GAPDH.


( F ) Immunofluorescence microscopy was performed on SH - SY5Y wild - type and Parkin KO cells either untreated or treated with AO for 2 hours . Cells were immunostained for TOM20 ( red ) , PDH E2 / E3bp ( blue ) , and LAMP1 ( green ) . Zoom insets represent 5x magnification . Circles indicate TOM20 MDVs positive for LAMP1 and arrowheads indicate TOM20 MDVs negative for LAMP1 . Scale bar represents 10 µm .

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(F) Immunofluorescence microscopy was performed on SH-SY5Y wild-type and Parkin KO cells either untreated or treated with AO for 2 hours. Cells were immunostained for TOM20 (red), PDH E2/E3bp (blue), and LAMP1 (green). Zoom insets represent 5x magnification. Circles indicate TOM20 MDVs positive for LAMP1 and arrowheads indicate TOM20 MDVs negative for LAMP1. Scale bar represents 10 µm.


( G ) Quantitation of TOM20 - positive MDV colocalisation with LAMP1 were performed across all treatment groups and represented as the % of TOM20 - positive MDVs colocalising with LAMP1 . Results represent 3 independent experiments ( 28 - 53 cells / condition ) and statistical significance was determined using a Two - way ANOVA ( Sidak ) .

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(G) Quantitation of TOM20-positive MDV colocalisation with LAMP1 were performed across all treatment groups and represented as the % of TOM20-positive MDVs colocalising with LAMP1. Results represent 3 independent experiments (28-53 cells/condition) and statistical significance was determined using a Two-way ANOVA (Sidak).


( H ) HeLa cells transiently transfected with HA - Parkin were subjected to mitochondrial stress by treatment with 25 μM antimycin A ( AA ) for 2 hours , then fixed and stained with antibodies specific to HA , PDH and TOM20 . TOM20 + ve / PDH - ve MDVs were quantified in cells that were either expressing or not expressing HA - Parkin ( n = 12 - 14 cells per condition from 3 independent experiments ) .

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(H) HeLa cells transiently transfected with HA-Parkin were subjected to mitochondrial stress by treatment with 25 μM antimycin A (AA) for 2 hours, then fixed and stained with antibodies specific to HA, PDH and TOM20. TOM20+ve/PDH-ve MDVs were quantified in cells that were either expressing or not expressing HA-Parkin (n = 12-14 cells per condition from 3 independent experiments).


( I ) Hela cells stably coexpressing BioID - Tollip and either HA - Parkin WT or Δ1 - 76 ( ΔUBL ) were incubated with and without biotin in the presence of AO for 6 hours prior to being subjected to cell lysis and streptavidin pulldowns . Western blot analysis was performed on lysate inputs and streptavidin pulldowns ( Streptavidin PD ) using antibodies against the indicated proteins or epitope tags .

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

(I) Hela cells stably coexpressing BioID-Tollip and either HA-Parkin WT or Δ1-76 (ΔUBL) were incubated with and without biotin in the presence of AO for 6 hours prior to being subjected to cell lysis and streptavidin pulldowns. Western blot analysis was performed on lysate inputs and streptavidin pulldowns (Streptavidin PD) using antibodies against the indicated proteins or epitope tags.


( A - B ) siRNA knockdown of Tollip or Parkin in SH - SY5Y cells was performed over a 72 hour period , then 18 hours prior to treatment cells were transfected with GFP - 2xFYVE to label endocytic membranes . Cells were treated with 25 μM antimycin A ( AA ) for 2 hours then fixed and immunostained with antibodies specific to PDH E2 / E3bp ( red ) and TOM20 ( blue ) . Arrowheads denote TOM20 + ve / PDH - ve MDVs that colocalise with GFP - 2xFYVE and arrows denote TOM20 + ve / PDH - ve MDVs that do not .

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(A-B) siRNA knockdown of Tollip or Parkin in SH-SY5Y cells was performed over a 72 hour period, then 18 hours prior to treatment cells were transfected with GFP-2xFYVE to label endocytic membranes. Cells were treated with 25 μM antimycin A (AA) for 2 hours then fixed and immunostained with antibodies specific to PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs that colocalise with GFP-2xFYVE and arrows denote TOM20+ve/PDH-ve MDVs that do not.


( C ) Quantification of TOM20 + ve / PDH - ve MDVs that are positive for GFP - 2xFYVE are shown as a % of TOM20 MDVs positive for GFP , with each data point indicating 1 cell . MDV colocalisation were quantified by eye from images captured ( n = 20 - 22 cells per condition from 3 independent experiments ) .

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(C) Quantification of TOM20+ve/PDH-ve MDVs that are positive for GFP-2xFYVE are shown as a % of TOM20 MDVs positive for GFP, with each data point indicating 1 cell. MDV colocalisation were quantified by eye from images captured (n = 20-22 cells per condition from 3 independent experiments).


( D ) SH - SY5Y cells were left untreated or treated with 25 μM AA for 2 hours prior to fixation and immunostaining for EEA1 ( green ) , PDH E2 / E3bp ( red ) , and TOM20 ( blue ) . Arrowheads indicate TOM20 + ve / PDH - ve MDVs positive for the early endosome marker EEA1 .

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(D) SH-SY5Y cells were left untreated or treated with 25 μM AA for 2 hours prior to fixation and immunostaining for EEA1 (green), PDH E2/E3bp (red), and TOM20 (blue). Arrowheads indicate TOM20+ve/PDH-ve MDVs positive for the early endosome marker EEA1.


( E ) siRNA knockdown of Parkin was performed in SH - SY5Y cells followed by 25 μM AA for 2 hours prior to fixation and immunostaining with antibodies specific to TOM20 ( green ) , Tollip ( red ) and Cytochrome c ( blue ) . We observed Tollip colocalisation with TOM20 + ve / PDH - ve MDVs ( denoted by arrowheads ) , which was still maintained following Parkin siRNA knockdown .

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(E) siRNA knockdown of Parkin was performed in SH-SY5Y cells followed by 25 μM AA for 2 hours prior to fixation and immunostaining with antibodies specific to TOM20 (green), Tollip (red) and Cytochrome c (blue). We observed Tollip colocalisation with TOM20+ve/PDH-ve MDVs (denoted by arrowheads), which was still maintained following Parkin siRNA knockdown.


( F ) SH - SY5Y cells were transfected with GFP - Tollip and treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or AO / 100 μM bafilomycin A ( BfnA1 ) for 2 hours prior to fixation and immunostaining with antibodies specific to GFP ( green ) , Rab7a ( red ) and PDH E2 / E3bp ( blue ) . ( G ) Quantification of GFP - Tollip / Rab7a colocalisation using Pearson ' s correlation .

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(F) SH-SY5Y cells were transfected with GFP-Tollip and treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 2 hours prior to fixation and immunostaining with antibodies specific to GFP (green), Rab7a (red) and PDH E2/E3bp (blue). (G) Quantification of GFP-Tollip/Rab7a colocalisation using Pearson's correlation.


( A ) Quantitation of Tollip vesicular clusters in SH - SY5Ys treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) for 2 hours . The percentage of cells with vesicular clusters were quantified from 7 - 23 cells for each condition from 4 independent experiments .

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(A) Quantitation of Tollip vesicular clusters in SH-SY5Ys treated with 5 μM antimycin A/10 μM oligomycin B (AO) for 2 hours. The percentage of cells with vesicular clusters were quantified from 7-23 cells for each condition from 4 independent experiments.


( B ) Quantitation of the prevalence of Tollip clusters in HEK293 WT or Tom1 KO cells treated with AO for 2 hours . The percentage of cells with vesicular clusters were quantified from an average of 50 cells for each condition from 5 independent experiments .

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(B) Quantitation of the prevalence of Tollip clusters in HEK293 WT or Tom1 KO cells treated with AO for 2 hours. The percentage of cells with vesicular clusters were quantified from an average of 50 cells for each condition from 5 independent experiments.


( C ) Endogenous Tollip and LAMP1 colocalise in SH - SY5Y cells treated with AO for 2 hours . Antibodies specific to LAMP1 ( green ) and Tollip ( red ) were used .

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(C) Endogenous Tollip and LAMP1 colocalise in SH-SY5Y cells treated with AO for 2 hours. Antibodies specific to LAMP1 (green) and Tollip (red) were used.


( D - E ) SH - SY5Y cells were transfected with either wild - type GFP - Tollip ( WT ) , GFP - Tollip lacking its N - terminus ( ∆ Nterm ) , or GFP - Tollip containing a CUE domain mutation ( CUEmut ) then treated with AO for 2 hours . Cells were fixed and immunostained with antibodies specific to GFP ( green ) and LAMP1 ( red ) .

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(D-E) SH-SY5Y cells were transfected with either wild-type GFP-Tollip (WT), GFP-Tollip lacking its N-terminus (∆Nterm), or GFP-Tollip containing a CUE domain mutation (CUEmut) then treated with AO for 2 hours. Cells were fixed and immunostained with antibodies specific to GFP (green) and LAMP1 (red).


( 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 .

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(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 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 ) .

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(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 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 .

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 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 B-EXP_ASSAY O B-SUBCELLULAR B-GENEPROD O B-GENEPROD O 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 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 O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O 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 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 .

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(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 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 ) .

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(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 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 .

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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 analysis of CD133 and GFAP using X08 GSCs and 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 analysis of CD133 and 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 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 .

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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.


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 .

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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 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 ) .

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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 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 .

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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 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 .

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

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


( 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 .

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(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 , 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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(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.


( A ) Immunoblotting of XO8 GSCs and DFCs .

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(A) Immunoblotting of XO8 GSCs and DFCs.


( 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 .

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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 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 .

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(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 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 .

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


( G ) Immunoblotting analysis of JNK and p38 .

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(G) Immunoblotting analysis of JNK and p38.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

B-CELL_LINE O O 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 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 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 O O

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


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

O O B-CELL_LINE 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 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 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 O O

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


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

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

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


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

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

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


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

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

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

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


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

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

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


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

O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O 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 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 O

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


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

O O B-CELL_LINE 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 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-GENEPROD 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 O O O O O O O O O O O

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


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

O O B-CELL_LINE 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 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-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

E. HEK293 cells were transfected with a pool of siRNA against CHK2 (siCHK2) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of CHK2, together with GSNOR were assessed by Western blot. GAPDH was used as loading control. Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.


F . HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 ( KU ) . Lysates were subjected to biotin - switch assay , and S - nitrosylated proteins ( PSNOs ) revealed by incubation with horseradish peroxidase ( HRP ) - conjugated streptavidin . Results obtained in the absence of ascorbate ( - ASC ) are shown as negative controls . Densitometry of each lane intensity is normalized to LDH , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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

F. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 (KU). Lysates were subjected to biotin-switch assay, and S-nitrosylated proteins (PSNOs) revealed by incubation with horseradish peroxidase (HRP)-conjugated streptavidin. Results obtained in the absence of ascorbate (- ASC) are shown as negative controls. Densitometry of each lane intensity is normalized to LDH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01.


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

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O 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 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 cells were treated for 8 or 24 h with 100 μM H2O2 or , alternatively , for 2 h with 2 mM hydroxyurea ( HU ) , selected as positive control of CHK1 phosphorylation at Ser317 . Phospho - CHK1Ser317 was assessed by Western blot ; densitometry normalized to Vinculin , selected as loading control , and expressed as arbitrary units ( with HU arbitrarily set to 1 ) . Values shown represent the means ± SD of n = 3 independent experiments . n . s . , not significant .

O O B-CELL_LINE O 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 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

H. HEK293 cells were treated for 8 or 24 h with 100 μM H2O2 or, alternatively, for 2 h with 2 mM hydroxyurea (HU), selected as positive control of CHK1 phosphorylation at Ser317. Phospho-CHK1Ser317 was assessed by Western blot; densitometry normalized to Vinculin, selected as loading control, and expressed as arbitrary units (with HU arbitrarily set to 1). Values shown represent the means ± SD of n = 3 independent experiments. n.s., not significant.


I . HEK293 cells were transfected with a pool of siRNA against CHK1 ( siCHK1 ) or with control siRNA ( siScr ) for 18 h and treated for additional 24 h with 100 μM H2O2 . CHK1 and GSNOR were assessed by Western blot analysis of . Densitometry of GSNOR immunoreactive bands is normalized to Vinculin , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 .

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

I. HEK293 cells were transfected with a pool of siRNA against CHK1 (siCHK1) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. CHK1 and GSNOR were assessed by Western blot analysis of. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.


A . HEK293 cells treated for 4 h with 100 μM H2O2 . Phospho - histone H2A . X ( γH2A . X ) was assessed by immunofluorescence analysis . Nuclei ( blue ) were stained with Hoechst 33342 . scale bar : 10μM . 4X digital magnification is shown at the bottom left of each image .

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

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

B. Additionally, γH2A.X was also assessed by Western blot at the indicated time points. GAPDH was used as loading control (top panel). Densitometry of each lane intensity is normalized to GAPDH, selected as loading control, and expressed as % of γH2A.X in H2O2-treated versus untreated cells. Values represent the means ± SD of two independent experiments (bottom panel).


C . HEK293 cells were treated for 8 or 24 h with 100 μM H2O2 or , alternatively , for 2 h with 2 mM hydroxyurea ( HU ) , selected as positive control of ATR / CHK1 axis activation . Basal and phospho - active forms of ATR and CHK1 ( at Ser345 ) were assessed by Western blot . GAPDH was used as loading control .

O O B-CELL_LINE O 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 O B-EXP_ASSAY O O O O B-GENEPROD O B-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 cells were treated for 8 h with 20 μM neocarzinostatin ( NCS ) . ( left ) Western blot analysis of basal and phospho - active forms of ATM , CHK2 , ATR , CHK1 , p53 , and GSNOR in HEK293 cells treated for 8 h with 20 μM neocarzinostatin ( NCS ) . Vinculin and GAPDH were used as loading controls . ( right ) Phospho : basal level ratios of ATM , CHK2 , ATR , CHK1 , along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

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

D. HEK293 cells were treated for 8 h with 20 μM neocarzinostatin (NCS). (left) Western blot analysis of basal and phospho-active forms of ATM, CHK2, ATR, CHK1, p53, and GSNOR in HEK293 cells treated for 8 h with 20 μM neocarzinostatin (NCS). Vinculin and GAPDH were used as loading controls. (right) Phospho:basal level ratios of ATM, CHK2, ATR, CHK1, along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.


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

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


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

O O B-CELL_LINE O O 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 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 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 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

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


F . HEK293 cells were transfected with plasmids coding for the wild - type ( WT ) , C2991L redox mutant ( CL ) of ATM , or with an empty vector ( Empty ) ( used as negative control ) for 40 h and treated for additional 8 h with 100 μM H2O2 . Basal and phospho - active forms of ATM , and GSNOR were assessed by Western blot analysis . Vinculin and GAPDH were used as loading controls . Two different exposures ( low and high ) were selected to highlight differences in P - ATM levels in different experimental settings .

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

F. HEK293 cells were transfected with plasmids coding for the wild-type (WT), C2991L redox mutant (CL) of ATM, or with an empty vector (Empty) (used as negative control) for 40 h and treated for additional 8 h with 100 μM H2O2. Basal and phospho-active forms of ATM, and GSNOR were assessed by Western blot analysis. Vinculin and GAPDH were used as loading controls. Two different exposures (low and high) were selected to highlight differences in P-ATM levels in different experimental settings.


A . HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of 20 μM pifithrin - α ( Pft ) . Basal and phospho - active forms of ATM , CHK2 , p53 , and GSNOR were assessed by Western blot . Phospho : basal level ratios of ATM , CHK2 and p53 ( normalized to Vinculin ) , along with densitometry of GSNOR immunoreactive bands ( normalized to GAPDH ) are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

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