Extended Data Fig. 1: Dissection of paused pluripotency in Mettl3−/− models.
From: m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency
a. m6A increase in paused ESCs was validated in an independent mass spectrometry experiment. Levels relative to control (Ctrl) for each replicate are shown (n = 3 biological replicates). b. Validation of Rptor and Rictor knockdown by RT-qPCR in paused ESCs grown in FBS/LIF/2i (n = 4 biological replicates). c. mTOR inhibition by Rptor and Rictor knockdown induces a paused phenotype with reduced cell proliferation and total RNA levels (n = 4 biological replicates). d. Dot blot showing an increase in m6A levels in ESCs upon knockdown of Rptor and Rictor. Levels of m6A are normalized to RNA loading control (methylene blue staining, n = 4 biological replicates). e. Design of Mettl3−/− ESCs models used in this study. f. Validation of Mettl3−/− ESCs, in control and pausing conditions, by western blot (representative of 3 biological replicates). g. Validation of Mettl3−/− #2–4 ESCs, in control and pausing conditions, by western blot (representative of 2 biological replicates). h. Mettl3−/− #2–4 ESCs also fail to suppress proliferation in paused conditions (n = 3 biological replicates). i. Mettl3-knockout mutant model in mice (Mettl3TCP−/−) and genotyping by PCR (left). Example of PCR genotyping of embryos resulting from Mettl3TCP+/− crossing, representative of all genotyping performed in this study [n(Mettl3TCP+/+) = 87, n(Mettl3TCP+/+) = 132, n(Mettl3TCP+/+) = 46]. +/+: wildtype, +/-: heterozygous, -/-: knockout. j. Validation of Mettl3TCP−/− in embryos by immunofluorescence. Representative staining images are shown. Number of embryos (n) as indicated. Scale bars = 30 µm. Data are mean ± SD (a-d) or mean ± SEM (h). P-values (as indicated on figure) by one-way ANOVA with Dunnett’s multiple comparison tests (a-c), two-tailed ratio paired Student’s t-tests (d), and linear regression test with interaction (h).
