FIGURE 5.

Expression of DmmtSSB variants impedes recovery from mtDNA depletion in Drosophila S2 cells. A, left panel, 0.2 μg/ml of EtBr was applied for 3 days to the growth media of cells carrying the DmmtSSBwt gene, followed by a recovery time of 12 days upon removal of EtBr. Control cells had neither dsRNA or CuSO₄ treatment (diamonds and solid line), whereas experimental lines were treated with dsRNA only (squares and solid line), dsRNA and 40 μm CuSO₄ (triangles and solid line), or dsRNA and 400 μm CuSO₄ (circles and dashed line). The time point in which each treatment was initiated is indicated with arrows. Relative mtDNA copy number was analyzed by qPCR, as described under “Experimental Procedures.” Right panel, DmmtSSB and β-tubulin protein levels were analyzed by immunoblot, as described under “Experimental Procedures.” B, the experimental design was as in A using cells expressing different DmmtSSB variant genes. The concentrations of CuSO₄ used to induce the overexpression and endogenous-level expression of DmmtSSB proteins are indicated. Cells were harvested at day 12 (9 days of recovery from EtBr treatment). DmmtSSB and β-tubulin protein levels were analyzed by immunoblot (upper panel), and relative mtDNA copy number was analyzed by qPCR (lower panel). The data were normalized to the ratio of mitochondrial/nuclear DNA in control S2 cells (arbitrarily set to 1), and represent the average of experiments with two independent cell lines. Error bars represent the standard deviation among the lines.