Fig. 3. S-glutathionylated but polysulfidated Drp1 prevents its activation after hypoxic stress.

A Timeline and schedule. After NRCMs were treated with GSSG or Na₂S₃, cells were washed to remove these, and then cultured under normoxia (Nor) or hypoxia (Hyp). B Effect of pretreatment of GSSG or Na₂S₃ on the GTP-binding activity of Drp1. (n = 3 independent experiments). C Effect of GSSG treatment on the GTP-binding activity of Drp1 wild-type (WT) or C644S (n = 3 independent experiments). D Representative images of GFP-Drp1 (green) and mitochondria (red) in NRCMs treated with or without GSSG under Nor or Hyp. GFP-Drp1 with or without myc-Grx1 was transfected into NRCMs. The dashed square is enlarged and is shown as green single channel. The average number of GFP-Drp1 particles per cell was quantified. (n = 3 independent experiments). Scale bars, 20 µm. E Effect of Grx1 expression on the inhibition of the GTP-binding activity by GSSG. (n = 3 independent experiments). F Polysulfidation (PolyS) of Drp1 under Nor or Hyp. (n = 3 independent experiments). G Effect of hypoxic stress on Drp1 S-glutathionylation. S-glutathionylated Drp1 was immunoprecipitated using an anti-glutathione antibody. (n = 4 independent experiments). Data are shown as the means ± SEM. Significance was determined by one-way ANOVA followed by Tukey’s post-hoc test or two-sided unpaired t-test F. Source data are provided as a Source Data file.