Immunofluorescence assay

For cell immunofluorescence assay, the PIG1 cells were cultured on laser confocal glass dishes (#801002, NEST Biotechnology, China) at 1.0 × 10⁵ cells per dish. After corresponding treatments, the cells were washed three times with phosphate buffer solution (PBS) and fixed with 4% paraformaldehyde for 10 min, followed by incubation with 0.1% Triton X-100 for 10 min and blocked with normal goat serum for 30 min. Then, cells were incubated with primary antibody (KEAP1 (D6B12) Rabbit mAb, 1:200, #8047, Cell Signaling Technology, USA; PGAM5 Rabbit mAb, 1:200, #ab126534, Abcam, UK; COX-IV Mouse mAb, 1:200, #ab33985, Abcam, UK) at 4 °C overnight. The corresponding secondary antibody (Goat anti-Rabbit IgG Green 488, 1:200, #ab150077, Abcam, UK; Goat anti-Mouse IgG Red Cy3, 1:200, #ab97035, Abcam, UK) was incubated for 1 h at room temperature in the dark. Then the cell nucleus was marked with the nuclear dye 4′,6-diamidino-2-phenylindole (DAPI) (1:1000, #ab104139, Abcam, UK) for 10 min at room temperature in the dark. Cells were washed three times with PBS after each step. The fluorescence was detected by laser confocal microscopy (#LSM510, Carl Zeiss AB, Germany). The quantitative analysis of co-localization between KEAP1 or PGAM5 and mitochondria was evaluated by calculating the ratio of KEAP1⁺COX IV⁺ dots or PGAM5⁺COX IV⁺ dots to COXIV⁺ dots. Three cells were randomly selected from each group and the experiment was duplicated three times for statistical analysis.

For skin specimens, paraffin-embedded 5-μm tissue sections were deparaffinized and heat-mediated antigen retrieval with Tris-EDTA buffer (PH = 9.0). Next, the skin sections were incubated with 5% goat serum (#AR0009, BosterBio, USA) for 30 min at room temperature. The remaining procedures were similar to the cell immunofluorescence assay with the use of an additional primary anti-Melan A (1:200, #ab187369, Abcam, UK) and the corresponding secondary Goat anti-Rabbit IgG Red Cy3 (1:200, #ab6939, Abcam, UK).