Fig. 5: Increased ribonucleotide incorporation into mtDNA from senescent cells.
From: Ribonucleotide incorporation into mitochondrial DNA drives inflammation
a, LC–MS analysis of the ratios of ribonucleotide and deoxyribonucleotide triphosphates from cell extracts of proliferating (Prol) and irradiation-induced senescent (Sen (IR)) IMR90 cells. n = 5 independent cultures. b, Untreated and RNase-H2-treated DNA isolated from proliferating and irradiation-induced senescent IMR90 cells was resolved on alkaline agarose gels and further analysed by Southern blotting using a Cox1 probe (left). Southern blots were analysed as described in Fig. 4d. A representative plot of n = 3 biologically independent experiments is shown. c, dPCR analysis of mtDNA levels in the cytosolic fractions of proliferating and irradiation-induced senescent IMR90 cells with and without deoxyribonucleoside supplementation. A probe/primer set for the D-loop region was used. n = 3 biologically independent experiments. d, Untreated and RNase-H2-treated mtDNA isolated from proliferating and irradiation-induced senescent IMR90 cells, which were supplemented with all four deoxyribonucleosides where indicated, was resolved on alkaline agarose gels and further analysed by Southern blotting and quantified as described in Fig. 4d. e, SASP gene expression was determined using quantitative PCR with reverse transcription (RT–qPCR) in proliferating and irradiation-induced senescent IMR90 cells, which were supplemented with deoxyribonucleosides where indicated. n = 3 biologically independent experiments. P values were calculated using unpaired two-tailed Student t-tests (a), one-way ANOVA with Tukey’s multiple-comparison test (c) or two-way ANOVA with Tukey’s multiple-comparison test (e). Data are mean ± s.d.
