Down-regulation of FAM92A1 caused defects in mitochondrial function. (A) FAM92A1 siRNA induced efficient silencing of FAM92A1. (B) Cell proliferation in glucose or galactose medium after depletion of FAM92A1 was determined using MTT assays. The cell growth inhibition at 120 h is shown in the bar graph. (C) Mitochondrial membrane potential was detected with TMRM staining. (D) Superoxide species in mitochondria were detected by MitoSOX. (E) Production of cellular ROS was detected by dihydroethidium. (F) OCR was measured using a Seahorse XF96^e Analyzer. (G) Statistical analysis of OCR in F. (H) Changes of the cellular ATP levels after FAN92A1 knockdown and rescue by WT FAM92A1. (I) Effects of FAM92A1 down-regulation on complex assembly of complexes I–V and rescue by WT FAM92A1. (J) Effects of FAM92A1 knockdown on the enzyme activities of complex I and IV and rescue by WT FAM92A1. The enzyme activity was normalized to the protein amount and the corresponding activity of citrate synthase (CS). (K) The steady-state level of respiratory chain components in FAM92A1-knockdown cells. In C–J, WT FAM92A1 was expressed in cells after 20 h siRNA treatment. Cells were harvested after 48 h expression of WT FAM92A1. Error bars indicate ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001, Student's t test.