Nutrient-Deprived Cell Death Assay

Most cell lines were obtained from the American Type Culture Collection and cultured in either DMEM or RPMI 1640 medium with 10% FBS (Gibco, Newcastle, Australia) and 100 U/ml penicillin/streptomycin in a 5% CO₂ incubator. HCC-M cells^{27 (link)} were kindly provided by Dr E.C. Ren (National University of Singapore). Nutrient levels in cell medium were restricted as previously described^{17 (link)}. After brief rinsing with PBS twice, the cells were starved in Earle’s balanced salt solution (with 1 g/L glucose) or dual glucose-free and glutamine-free DMEM (Thermo-Fisher). Cell death and survival were measured by subG1, propidium iodide (PI, Sigma‒Aldrich) exclusion assay or lactate dehydrogenase (LDH) assays^{17 (link)}. Transient silencing was performed using Lipofectamine RNAiMAX transfection reagent with siRNAs specific for several known E3 ubiquitin ligases involved in mitophagy (PINK1, SIAH1, STUB1, and MUL1), all obtained from Thermo-Fisher.