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. 2018 May 31;46(13):6642–6669. doi: 10.1093/nar/gky456

Figure 10.

Figure 10.

De novo replication is affected in heart Sod2 knockout mice. (A) Representative experiment of in organello replication assay in heart Sod2 loxP x Ckmm cre mice. The mtDNA was radioactively labeled in isolated mitochondria, purified and half of it was boiled to release newly synthesized 7S DNA. Samples were separated on an agarose gel and transferred to a membrane. Small aliquot representing the input was analyzed with Coomassie staining after the labeling. (B) Quantification of de novo replication, the relative incorporation of radioactivity into mtDNA and newly synthesized 7S DNA in Sod2 loxP x Ckmm cre mice. The incorporation was normalized to steady-state level of mtDNA that was probed from the same membrane after the de novo signal could not be detected anymore. White circles indicate samples from controls (pp, n = 9, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 9, 9–10 week old). (C) Representative experiment of in organello transcription assay from Sod2 loxP x Ckmm cre mice. The mtRNA was radioactively labeled in isolated mitochondria and half of the sample was purified (pulse). Another half was incubated with non-radioactive UTP for 2 h (chase) and purified, and then samples were analyzed with northern blotting. Small aliquot representing the input was analyzed with Coomassie staining after the labeling. (D) Quantification of de novo transcription, the relative incorporation of radioactivity into mtRNA in Sod2 loxP x Ckmm cre mice. The incorporation was normalized to steady-state level of CytB that was probed from the same membrane after the de novo signal could not be detected anymore. See Supplementary Figure S7, which shows that CytB levels do not change in Sod2 loxP x Ckmm cre mice. White circles indicate samples from controls (pp, n = 12, 10–11 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 12, 9–10 week old). Horizontal lines represent means, error bars represent SD, ****P< 0.0001, Student's t-test.