Fig. 5: C15 and C42 are functionally essential for ROMO1-mediated thiol-protection.

a Non-reducing SDS-PAGE analysis for disulfide-bonded homodimer formation of WT and mutant ROMO1. Purified human WT or mutant ROMO1-FLAG protein was oxidized with 10 µM H₂O₂. CBB staining was used. b Anti-ROMO1 western blots showing formation of DTT-sensitive high molecular weight complexes in neonatal rat cardiomyocytes overexpressing WT or C15S, C27S, C42S, or C79S mutant ROMO1. Anti-ATP5A served as the internal control. c S-sulfinic oxidation levels induced by 200 μM H₂O₂ treatment in neonatal rat cardiomyocytes overexpressing WT, or C15S, C27S, C42S, or C79S mutant ROMO1. CBB staining served as the internal control. d Iodoacetamide-alkyne labeling showing elevated oxidation of the mitochondrial cysteinome in the C15S or C42S conditional knockin hearts (C15S cKI or C42S cKI). The reactive thiols of the mitochondrial cysteinome were labeled with iodoacetamide-alkyne and then reacted with azide-biotin tag via click chemistry. CBB staining served as the internal control. e, f Effects of WT or mutant ROMO1 overexpression (OE) on protecting against oxidative stress (1 mM H₂O₂ treatment) induced mPTP opening (e) and cell death (f) in adult rat cardiomyocytes. mPTP opening was indicated by ΔΨ_m loss and cell death was assessed by lactate dehydrogenase (LDH) release. Data are mean ± s.e.m. In e, n = 59 dishes from 5 independent experiments for Control, n = 20 dishes from 5 independent experiments for WT OE, n = 25 dishes from 5 independent experiments for C15S OE, n = 30 dishes from 5 independent experiments for C27S OE, n = 24 dishes from 5 independent experiments for C42S OE, n = 30 dishes from 5 independent experiments for C79S OE. In f, n = 20 measurements from 5 independent experiments for Control, n = 5 measurements from 5 independent experiments for WT, C15S, C27S, C42S, and C79S OE. g Representative western blots showing comparable expression of WT and C15S, C27S, C42S, or C79S mutant ROMO1-FLAG in HeLa cells. Anti-ATP5B served as the internal control. h Effects of overexpressing WT or mutant ROMO1 on mitochondrial Ca²⁺ uptake induced by 100 µM histamine stimulation in HeLa cells. Data are mean ± s.e.m. n = 12 dishes from 3 independent experiments for Control, n = 15 dishes from 3 independent experiments for WT OE, n = 6 dishes from 3 independent experiments for C15S OE, n = 5 dishes from 3 independent experiments for C27S OE, n = 10 dishes from 3 independent experiments for C42S OE, n = 5 dishes from 3 independent experiments for C79S OE. Data in a–d, g are representative of three independent experiments. In e, f, h, two-tailed unpaired Student’s t-test with Welch’s correction was used. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance. Source data are provided as a Source Data file.