Extended Data Fig. 4: Modulating mitochondrial activity affects 5mC levels.

a, Anti-5mC immunofluorescence on S3+/+ cells in 2i or 2iLIF and S3-/- cells in 2iLIF treated with EdU for 4 h. Violin plots show the distribution of an average of 67 nuclei per sample; one representative experiment is shown for each condition. b, Anti-5mC immunofluorescence on S3+/+ cells treated with Rotenone or Antimycin A. Violin plots show the distribution of an average of 74 nuclei per sample. 3 independent experiments shown as individual violins. c, Anti-5mC immunofluorescence on E14 in 2iLIF and 2i, and on Dnmt3a/b double KO cells in 2iLIF, treated with Vehicle, Rotenone or Antimycin A. Violin plots show the distribution of an average of 183 nuclei per sample. 3 experiments shown as individual violins. d, Confocal images of S3+/+, S3-/- cells and MitoS3.A/B clones stained with anti-Stat3 and anti-Atad3 antibodies. Representative images of 3 independent experiments. e, Electron Microscopy images of STAT3 protein stained by Diaminobenzidine photooxidation method, in S3-/- and MitoS3.A cells. Representative images of 2 experiments. M, mitochondria; N, nucleus. f, Expression analysis of Socs3 in S3+/+, S3-/- cells and MitoS3.A/B clones in 2iLIF. Bars: mean of n = 2 experiments, shown as dots. g, (Left) Western blot of S3+/+, S3-/- cells and MitoS3.A and MitoS3.B clones in 2iLIF. LAMIN B: loading control. (Right) Western blot of total lysates or mitochondrial and nuclear fractions. The nuclear protein LAMIN B and mitochondrial marker TIM23 confirmed successful nuclear and mitochondrial isolation. Representative images of 2 independent experiment. h, Oxygen consumption rate measured by Seahorse extracellular flux assay of S3+/+, S3-/- and MitoS3.A/B clones cultured in 2iLIF. Mean and S.D. of n = 5 biological replicates is shown. All violin and boxplots indicate the 1st, 2nd and 3rd quartiles, with whiskers indicating minimum and maximum value. All p-values calculated by two-tailed unpaired T-test. Scale bars: 20μm.

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