Figure 3

Chloramphenicol selection for transferred CAP-R mtDNA retention. (a) Selection of mouse ρ0 or ρ + mutant cells with successfully retained exogenous CAP-R 501-1 mtDNA. (b) Cell lines used with known nuclear and mitochondrial mouse backgrounds. (c) Seahorse Extracellular Flux analysis quantification of basal and maximal cellular respiration in Δmt-ND4, Δmt-ND6, CAP-R 501-1, and L929 ρ0 cells. Two-tailed, unpaired Student’s t-test comparing samples to L929ρ0. * represents significance with * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Black * represents significance for Basal Respiration and Blue * represents significance for Maximal Respiration. The bar height denotes average of 3 replicates and the error bars are the standard deviation. (d) Phosphate buffered saline (PBS) or CAP-R 501-1 mitochondria were transferred into L929 ρ0, Δmt-ND4, and Δmt-ND6 recipient cells and were selected on uridine-deficient, CAP-supplemented media. Four weeks after mitochondrial transfer, colonies were imaged with an inverted microscope and 5 × objective. Scale bar denotes 100 µm. (e) RFLP analysis of CAP-R 501-1, L929 ρ0, L929 ρ0 + CAP-R 501-1, Δmt-ND4 + CAP-R 501-1, and Δmt-ND6 + CAP-R 501-1 bulk culture cells two weeks after mitochondrial transfer. (f) Following Δmt-ND4 + CAP-R 501-1 mitochondrial transfer, cells were cultured in (1) uridine-supplemented media for four days, (2) uridine-deficient, CAP-supplemented media for 24 days, and (3) uridine-supplemented media with or without CAP for 7 days. RFLP analysis of CAP-R 501-1, Δmt-ND4, and Δmt-ND4 + CAP-R 501-1 mitochondria. In (e–f), arrows denote the difference between CAP-S (502 bp) and CAP-R (434 bp) PCR products post-MaeII digestion on a 2.5% agarose gel electrophoresis. CAP-R 501-1 control is the same in each panel. Each of these panels were cropped from different parts of the same gel with the same exposure level.