Fig. 4
From: CO2-sensitive tRNA modification associated with human mitochondrial disease
Mitochondrial dysfunction in OSGEPL1 KO cells. a Growth curves for WT HEK293T, OSGEPL1 KO#1, and OSGEPL1 KO#2 cells cultured in the presence of glucose (left) or galactose (right) as the primary carbon source. Mean values ± s.e.m. of four independent cultures are plotted. b Oxygen consumption rates of WT, OSGEPL1 KO#1, and OSGEPL1 KO#2 cells measured using an XFp extracellular flux analyzer. Mean values ± s.d. of biological triplicates are compared. *P < 0.05, Student’s t-test. c Steady-state levels of ATP in WT, OSGEPL1 KO#1, and OSGEPL1 KO#2 cells. Mean values ± s.e.m. of three independent experiments are compared. *P < 0.05, Student’s t-test. d Relative activities of respiratory complexes I, II, III, and IV (CI–IV) in WT, OSGEPL1 KO#1, and KO#2 cells. Mean values ± s.e.m. of three independent experiments are compared. *P < 0.05, Student’s t-test. e Steady-state levels of subunit proteins in respiratory chain complexes. Mitochondrial fractions of WT, OSGEPL1 KO#1, and KO#2 cells resolved by SDS-PAGE were analyzed by western blotting with the indicated antibodies. A gel stained with Coomassie brilliant blue (CBB) is shown in Supplementary Fig. 6a. Uncut gel images are provided in Supplementary Fig. 15. f Pulse labeling of mitochondrial protein synthesis. WT, OSGEPL1 KO#1, and KO#2 cells were labeled with [35S] methionine and [35S] cysteine after cytoplasmic protein synthesis was halted with emetine. Whole-cell lysates were resolved by Tricine-SDS-PAGE and stained with CBB as a loading control (Supplementary Fig. 6b). The dried gel was exposed to an imaging plate and visualized on a fluorimager. Assignment of mitochondrial proteins is indicated. Uncut gel images are provided in Supplementary Fig. 15. g In vivo aminoacylation levels of mt-tRNALys and mt-tRNAVal in WT, OSGEPL1 KO#1, and OSGEPL1 KO#2 cells. Crude aminoacyl-tRNAs extracted under acidic conditions were treated with (+) or without (−) mild alkali for deacylation and subjected to acid urea-PAGE and northern blotting. White and black arrowheads indicate aminoacyl-tRNA and deacyl-tRNA, respectively. Aminoacylation level of each condition was calculated from the band intensities. Lysylation levels of mt-tRNAsLys from WT, KO#1, and KO#2 are 98.4%, 89.9%, and 83.9%, respectively. Valylation levels of mt-tRNAsVal from WT, KO#1, and KO#2 are 90.1%, 90.3%, and 89.1%, respectively. Uncut gel images are provided in Supplementary Fig. 15
