Human mtDNA-depleted (r 0 ) 143B TK 2 osteosarcoma cells and parental normal r + cells (provided from Dr. Wei YH, National Yang-Ming Univ., Taipei, Taiwan) were cultured in high glucose (4.5 g/L) Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), antibiotics (100 mg/ml penicillin/streptomycin mix), and uridine (50 mg/ml) in a humidified atmosphere at 37uC with 5% CO 2 as described [49] . The absence of mtDNA in r 0 cells was verified by PCR and PicoGreen staining ( Figure S4 ). PicoGreen is a sensitive staining dye to visualize mtDNA [50] . Pure mitochondria from r 0 and r + cells (2610 8 ) were prepared by ultracentrifugation by using 30-50% (1.1 and 1.6 g/ml) Optiprep TM density gradient media (Sigma-Aldrich) as described previously [51] . The purity of mitochondria was confirmed by western blot analysis. Mt proteins, COX I, COX IV, and HSPD1 (HSP60), exhibited significant reductions in levels in r 0 mitochondria compared to their levels in r + mitochondria, supporting complete mtDNA depletion and good mt preparation. Meanwhile, nuclear fraction marker (HDAC1), cytoplasmic fraction marker (NF-kB and SOD1), ER fraction marker (GRP78), and lysosomal marker (LAMP1) were not detected in both r 0 and r + mitochondria ( Figure S5) . b-actin was used for equal loading verification. Then, we applied quantitative cICAT analysis to compare protein abundances in r 0 and r + mitochondria.