Figure 5. ATM/GSNOR axis drives mitophagy upon H₂O₂ treatment.

• 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 was transfected for 48 h with siRNA against ATM (siATM), GSNOR (siGSNOR), or control siRNA (scramble, siScr). Afterward, 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), and MTCO2 (complex IV)]. Basal and phospho‐ATM and GSNOR were used to check the efficiency of siRNA‐mediated knockdown. Vinculin was used as loading control.
• D
  Densitometry of mitochondrial protein immunoreactive bands of panel C (normalized to Vinculin) is indicated as H₂O₂‐treated versus untreated cells (CTR) and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.
• E
  In the same experimental settings, mitophagy was also assessed by RT–qPCR relative quantitation of D‐loop (selected as measure of mtDNA) normalized to genomic actin (gActin). Results shown are the means ± SD of n = 8 experiments ***P < 0.001; n.s., not significant.
• F
  U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATM^WT, ATM^2RA, or ATM^CL 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.
• G, 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. 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 (25^th‐75^th 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.

Source data are available online for this figure.