Validation and characterization of selected low-ATP hits in respiratory conditions.

(A) K562 lines were generated expressing the dCas9-KRAB, Clover-mApple ATP FRET sensor, and 1 sgRNA against MRPL10 or non-targeting control guides (Cont1, Cont2), and ATP levels were assessed by flow cytometry. Compared with the ATP level in basal conditions, MRPL10 knockdown decreased ATP to a greater extent in respiratory conditions (box and whisker plots; line = median; box = 25th–75th percentile; whisker = 5th–95th percentile). In contrast, in glycolytic conditions, ATP levels dropped similarly in MRPL10 and control groups. N = 3,128–11,971 cells examined per group for FACS. ***p MRPL10 in K562 cells also decreased ATP levels by luciferase to a greater extent in respiratory conditions. In contrast, in glycolytic conditions, ATP levels again dropped similarly in MRPL10 and control groups. Data show mean ± SEM; N = 4 wells per group from 2 independent experiments for luciferase. **p p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Error bars are mean ± SEM; N = 10–32 wells/group, compiled from 2–4 experiments per cell line. (D, E) OCR (aerobic respiration rate) and ECAR (a surrogate of glycolysis) in K562 lines were measured using a 96-well Seahorse Extracellular Flux Analyzer. Arrows show the additions of the mitochondrial uncoupler FCCP (1 μM), the ATP synthase inhibitor oligomycin (1 μM), or the mitochondrial complex I inhibitor rotenone (1 μM). Knocking down of COX11, KPNB1, PDSS1, PDSS2, or ATP5MPL significantly decreased maximal respiration (after FCCP). Stars in legend box indicate significant difference of maximal respiration after FCCP (panel D). Oligomycin and rotenone additions similarly decreased OCR in all lines (panel E), indicating similar fractions of basal oxygen consumption devoted to mitochondrial respiration (oligomycin) and nonmitochondrial consumption (rotenone). Data are compiled from 4 experiments; n = 6–18 wells per group. Error bars represent SEM. NS, p > 0.05; *p p p S1 Data. Cont, control; dCas9, dead CRISPR-associated protein 9; ECAR, extracellular acidification rate; FACS, fluorescence-activated cell sorting; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FRET, fluorescence resonance energy transfer; KRAB, Kruppel-associated box; NS, not significant; OCR, oxygen consumption rate; sgRNA, single guide RNA.