Fig. 4: Increased ribonucleotide incorporation into mtDNA in Yme1l−/− MEFs and Mgme1−/− kidneys.
From: Ribonucleotide incorporation into mitochondrial DNA drives inflammation
a,b, LC–MS analysis of the ratio of rNTPs (rA, rG, rC, rU) and dNTPs (dA, dG, dC, dT) from cell extracts of Mgme1+/+ and Mgme1−/− (a) or Yme1l+/+ and Yme1l−/− (b) immortalized MEFs. n = 6 independent cultures. c, HydEn-seq analysis of mtDNA isolated from Yme1l+/+ and Yme1l−/− immortalized MEFs. Each ribonucleotide is shown as the fraction of the total ribonucleotide content in the heavy strand (top) and light strand (bottom). n = 6 independent cultures. d, Alkaline agarose gel analysis of mtDNA isolated from Yme1l+/+ and Yme1l−/− immortalized MEFs, treated with RNase H2 as indicated and analysed using Southern blotting. mtDNA was visualized using a Cox1 probe (left). Southern blots were quantified and visualized using MultiGauge and Instant Clue software, respectively. The signal intensities of individual pixels were determined to generate plots for untreated (top) and RNase-H2-treated samples (bottom). The intensity data were scaled between 0 and 1. A representative plot of n = 3 biologically independent experiments is shown. e, HydEn-seq analysis of mtDNA isolated from Mgme1+/+ and Mgme1−/− immortalized MEFs as described in c. n = 4 independent cultures. f, Untreated and RNase-H2-treated mtDNA isolated from control and Mgme1−/− kidney tissue was resolved on alkaline agarose gels and analysed by Southern blotting using a Cox1 probe (left). Densitometry plots of the Southern blots were normalized as described in d (right) and aggregated using the average normalized intensity of an individual replicate. A representative plot containing two biological replicates of n = 3 technical replicates is shown. P values were calculated using unpaired two-tailed Student t-tests (a,b) and two-way ANOVA with Šidák’s multiple-comparison test (c,e). Data are mean ± s.d.
