Mitochondrial translation is dependent on membrane potential. (A) HEK293 cells were metabolically labeled with ³⁵S for a 45-min pulse and separated by SDS-PAGE. Cells were treated under a variety of conditions: DMSO (−); actinonin (Act) only during the pulse; pretreating with actinonin for 6 h before the pulse (6h Pre Act); 1-µM valinomycin (Val); 2.5-µM nigericin (Nig); and 10-µM CCCP. (B) Scatter plot of flow cytometry analysis with TMRM staining of HEK293 cells treated for 45 min with CCCP and valinomycin, and for 3 and 6 h with actinonin relative to the signal in untreated cells (control). All data originate from independent experiments. (C) Metabolic labeling with ³⁵S for 45 min in HEK293 cells with a titration of valinomycin from 0 to 1 µM. (D) Corresponding flow cytometry analysis with TMRM staining of HEK293 cells treated for 45 min with valinomycin from 0 to 1 µM. Quantification from three independent experiments. (E and F) The relationship between translation performance (relative signal intensity from metabolic labeling) and membrane potential (mean TMRM intensity) for valinomycin (E) and CCCP (F). The data are representative of three independent experiments.