Figure 2.

Short, 3-day knockdown of Twinkle, mtSSB and GRSF1 does not fully deplete mtDNA copy-number. Because the primary interest of this paper was the possible link between RNA granules and mtDNA replication factors Twinkle and mtSSB, we had to make sure that knockdown of these proteins would not completely deplete mtDNA copy-number as that would also severely affect mtRNA synthesis. For this reason, we tested a short 68–72 h knockdown of Twinkle, mtSSB, GRSF1 and combinations thereof in three biological repeats in U2OS cells, isolated total cellular DNA and measured copy number using qPCR. Results are presented as relative copy-number compared to control cells treated in parallel with non-targeting siRNA. Error bars show +/- SEM. Statistics used one sample t-test compared each sample to its own control that was set at 100. * indicates a P-value ≤ 0.05; ** indicates a P-value ≤ 0.01. Results show that indeed the effect of a 3-day knockdown of the various proteins and their combinations did not result in a severe loss of mtDNA. In all subsequent experiments we therefore used a 68–72 h knockdown regime.