Fig. 5. ABHD10 modulates mitochondrial redox buffering capacity by regulating S-palmitoylation of PRDX5 active site.

(a) ABE assay in HEK293T cells demonstrates that PRDX5 S-palmitoylation level increases upon ABHD10 knockdown, whereas no changes are observed for PRDX3 and ALDH6A1. Calnexin (CANX) is shown as a stable S-palmitoylated protein control. (b) Quantification of relative S-palmitoylation levels of PRDX3 and PRDX5 upon ABHD10 knockdown as shown in a. Statistical analyses performed with a two-tailed Student’s t-test with unequal variance (n = 5 biological replicates). Data expressed as mean ± s.e.m. (c) Protein map showing the three cysteine residues in mature PRDX5. (d) ABE assay in HEK293T cells expressing various PRDX5-Flag constructs (WT and three Cys to Ser mutants) shows that S-palmitoylation occurs at the catalytic site Cys100. Two biological replicates performed. (e) HEK293T cells transfected with either NT siRNA or ABHD10 siRNA were challenged with varying concentrations of H₂O₂ and then analyzed for viability by the MTS assay. (f) HEK293T cells were transfected with either NT siRNA or ABHD10 siRNA were challenged with varying concentrations of paraquat and then analyzed for viability by the MTS assay. Statistical analyses performed with a two-tailed Student’s t-test with unequal variance (n ≥ 6 biological replicates in e and f). Data expressed as mean ± s.e.m. and cells with various concentrations of H₂O₂ (e) or paraquat (f) is normalized to respective cells treated with NT or ABHD10 siRNA and H₂O. Dots represent individual data points.