Fig. 1. Connections between S-depalmitoylation and mitochondrial redox buffering capacity.

(a) Representative images showing that PalmB treatment diminishes the mitochondrial redox buffering capacity as measured by mitoPY1 (mitochondrially targeted dye that generates H₂O₂-dependent fluorescence) in HEK293T cells when exposed to H₂O₂. 25 μm scale bar shown. (b) Quantification of the relative fluorescence intensity from mitoPY1 in each set of conditions shown in a. Statistical analyses performed with a two-tailed Student’s t-test with unequal variance (n = 6 images). Data expressed as mean ± s.e.m. and normalized to control cells treated with DMSO. Dots represent individual data points. (c) ABE assay confirms S-acylation of PRDX3 and PRDX5 in HEK293T cells, with calnexin (CANX) as a known S-palmitoylated protein control. Input: total protein before enrichment; Output: S-acylated protein enriched via biotin labeled thiols upon hydroxyl amine (HA) treatment which selectively cleaves thioester bonds to expose acylated thiols. (d) Metabolic labeling with the palmitic acid analogue, 17-ODYA (50 μM) further validates PRDX3 and PRDX5 are S-palmitoylated in HEK293T cells. 17-ODYA is metabolically incorporated into palmitoylated proteins and is clicked with biotin-azide for enrichment. The signal difference between −HA and +HA lanes indicates the palmitoylation modification occurs on cysteine residues. (e) PRDX3 and PRDX5 are prone to S-acylation in vivo as determined by an ABE assay performed on mouse tissues. (f) Pan-APT inhibitor PalmB treatment increases the S-palmitoylation level of PRDX3 and PRDX5 as determined by an ABE assay in HEK293T cells. Two biological replicates performed for b-d and f.