Loss of PUS1 impairs mitochondrial function in iPSCs. (A-B) Mitochondrial biomass (A) and the ratio of biomass to MMP (B) were evaluated in 3 iPSC lines by flow cytometry. The representative histogram (left) and geometric mean fluorescent intensity (gMFI, right) are shown. Normal, n = 3; MLASA, n = 2; MLASA-Res, n = 3. (C) Quantitative analysis of mtDNA copy number via quantitative reverse transcription polymerase chain reaction (qRT-PCR) in iPSCs (n = 4). (D) Cellular ATP levels in iPSCs detected by CellTiter-Glo 2.0 reagent (n = 3). (E-G) Mitochondrial (E), total (F), and cytoplasmic (G) ROS levels of iPSCs were evaluated by MitoSOX, H2DCFDA, and CellROX, respectively. The representative histogram (left) and gMFI (right) are shown (n = 3). (H-I) Measurement of cellular oxygen consumption in iPSCs. Oxygen consumption rates (OCRs) were monitored by injecting 1 μM oligomycin (Oligo), 0.5 μM FCCP, and 1μM rotenone/antimycin A (Rot/AA) in sequential order using the Seahorse XFe24 Extracellular Flux Analyzer (H). The average basal and maximum respirations were normalized with Normal-iPSCs (I; n = 3). (J) Activity analyses of mitochondrial respiratory chain complexes. Complex I, II, III, IV, and V activities were measured according to the manuals of relevant kits (n = 3). Values in all panels denote mean ± SD (∗P < .05; ∗∗P < .01; ∗∗∗P < .001); 1-way ANOVA. B2M, beta-2-microglobulin; FCCP, trifluoromethoxy carbonylcyanide phenylhydrazone; MT-LEU, mitochondria-tRNALeu.
