Epicm a

Mouse renal tubular epithelial cells (EpiCM-a) (Sciencell, San Diego, California, US) were thawed in a 38°C water bath and then centrifuged at 1,000 rpm for 5 min. The supernatant was discarded and the cells were cultured in DMEM medium in an incubator containing 5% CO₂ at 37°C. The medium was replaced when cells adhered to the bottle wall. The cells were subcultured until 80% of the bottle bottom was covered with cells. To induce hypoxia, mouse renal tubular epithelial cells were incubated in serum-free DMEM in a hypoxia chamber containing 95% N2 and 5% CO₂ at 37°C. Following exposure to hypoxic conditions, cell medium was replaced with fresh oxygenated DMEM and cells were reoxygenated for 24 h in normoxic conditions (5% CO², 21% O₂ and 74% N₂) at 37°C.The cells with traces of APPL1 siRNA treatment were divided into six groups: Sham group, HR group, HR+Curcumin (25 μM) group, HR+Curcumin+APPL1 siRNA group, HR+Curcumin+NC siRNA group, and HR+Curcumin+APPL1 siRNA+AZD5363 (30 μM) group. HR treatment consisted of hypoxia for 3 h and then reoxygenation for 24 h. Curcumin and AZD5363 were administered at the beginning of HR treatment.

Mouse renal tubular epithelial cells (EpiCM‐a)(Sciencell, San Diego, California, US) were thawed in a 38°C water bath and then centrifuged at 200 g for 5 minutes. The supernatant was discarded, and the cells were cultured in DMEM medium in an incubator containing 5% CO₂ at 37°C. The medium was replaced when cells adhered to the bottle wall. The cells were subcultured until the cells covered 80% of the bottom of the bottle. Cells were pre‐treated for 1 hours with 5Z‐7‐oxozeaenol dissolved in dimethyl sulfoxide (10 μmol/L in DMSO) or 3‐MA (10 μmol/L) according to the manufacturer's recommendation. Cells were treated with 10 μg/mL cisplatin or sham control for 6 hours.

The primary renal proximal tubular cells were extracted from male C57BL/6J mice (3 weeks old) (Kamiyama et al., 2012 (link)). Cells were seeded in 6 or 12-well plates as needed and cultured in Epithelial Cell medium (EpiCM-A; ScienCell, United States) supplemented with 2% fetal bovine serum (FBS; ScienCell), 1% penicillin-streptomycin (ScienCell) and 1% EpiCGS-a (ScienCell) in 37°C in an incubator containing 5% CO₂ and 95% humidified air. When reaching approximately 70% confluence, the medium was replaced, and the cells were exposed to different stimuli reagents for 24 h, including 30 mmol/L d-glucose (HG) (G8270, Sigma Aldrich), D-mannitol (MAN) (M4125, Sigma Aldrich) and dimethyloxallyl glycine (DMOG) (3695; Sigma-Aldrich, St Louis, United States).