Fig. 2: mitoARCUS effect on heteroplasmic cells carrying the tRNAAla mutation (m.5024C>T). | Nature Communications

Fig. 2: mitoARCUS effect on heteroplasmic cells carrying the tRNAAla mutation (m.5024C>T).

From: Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo

Fig. 2

a Example of FACS cell sorting gating. Cells were sorted by the presence of GFP co-expression: “Black” cells (bottom gate) and “Green” cells (top gate). b RFLP-HOT PCR analysis of two independent transfections and cell-sorting experiments of heteroplasmic cells carrying 50% m.5024C>T mutation. Mutant levels in the Green cell populations (Gr) were compared to Untransfected cells (U). This experiment was done once. c Quantification of heteroplasmy shift from the two cell-sorting experiments in cells carrying 50% mutation described in b. Results were compared to Untransfected cell heteroplasmy. d RFLP “last-cycle hot” PCR analysis of heteroplasmic cells carrying high heteroplasmic mutant load (90%) transfected with mitoARCUS over time. This experiment was repeated three times with similar results. e Quantification of Fig. 2d. Values are normalized to Untransfected cells. Black cells are named Blk (n = 4). f Total mtDNA levels were checked in highly mutant cells transfected with mitoARCUS and compared to untransfected cells 24 h after transfection, and followed for 3 weeks after transfection (n = 3). p values are related to untransfected cells (100%). g Oxygen consumption rate (OCR) was deduced in cells carrying high levels of heteroplasmic mutant mtDNA that were transfected with mitoARCUS and grown for 3 weeks [n = 3 (CTRL), n = 7 (Unt), n = 3 (Blk), n = 7 (Gr)]. Data are mean ± SEM. Statistical analysis was performed using two-tailed Student’s t-test.

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