Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress (2020)
Figure 1

Figure 1. GSNOR is translationally induced by hydrogen peroxide.

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. 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. 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. F, G. 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), or 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. 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.