Figure 2.

ΔmtDNA heteroplasmy fluctuations after induced mtDNA depletion. (A) An osteosarcoma cell line containing high levels of a ΔmtDNA was treated with 50 ng/ml EtBr for 15 days and allowed to recover for 30 additional days. Cells were harvested at the indicated times and their DNA purified and analyzed for relative levels of mtDNA (mutated or wild-type, closed and open circles, respectively) / nDNA as described in Materials and Methods. (B–D) Three cell lines harboring ΔmtDNA were subjected to a 15 day EtBr treatment followed by a 30 day recovery period. Parallel cultures growing in the same medium, but without EtBr were also analyzed to assess heteroplasmy fluctuations related to time in culture. Total DNA extracted at different times was analyzed by Southern blot and the percentage mutated mtDNA represented as filled triangles in the EtBr treated series and as open triangles in the control series. The mtDNA levels during EtBr treatment (days 7 and 15) were very low and did not allow a reliable estimation of heteroplasmy. There was a reduction in the percentage mutated mtDNA during normal growth conditions, but EtBr treatment was consistently associated with an increase in percentage mutated mtDNA immediately after the treatment.