Expression of CLPB mutants impairs mitochondrial respiration without affecting membrane potential. MOLM-13 cells were transduced with a lentiviral vector expressing WT CLPB, CLPB-N496K, or CLPB-R620C. (A) Representative photomicrographs of cells stained with anti-HA antibody to detect CLPB (red) and anti-TOM20 (yellow) to label mitochondria. Nuclei were counterstained with SYTOX Green (blue); original magnification, ×63. (B-E) The mitochondrial stress test was performed using a Seahorse XFe96 Analyzer. (B) Representative graph showing the oxygen consumption rate (OCR) at baseline and after treatment with the ATP synthase inhibitor oligomycin (OM) and then the uncoupling agent carbonyl cyanide-4 phenylhydrazone (FCCP). Parameters of mitochondrial respiration include basal respiration (C), maximal respiration (D), and total ATP production (E). (F-H) Cells were labeled TMRM or MitoTracker Green. (F) MitoTracker Green signal normalized to empty vector control. (G) TMRM signal normalized to empty vector control. (H) Ratio of TMRM signal/MitoTracker green signal. Mitochondrial respiration parameters for additional CLPB variants: basal respiration (I), maximal respiration (J), and total ATP production (K). *P < .05, **P < .01, repeated-measures 1-way ANOVA.