Fig. 1: mTORC1 inhibition only partially rescues Pten KO phenotype.

a Schematic representation of our experimental setup. Nx indicates differentiation time (x) after 2i withdrawal in N2B27 medium. b Flow cytometry analysis of Rex1-GFP levels in WT and two independent Pten KO clones in naïve pluripotency supporting conditions (2i, green) and 24 h after 2i removal (N24, blue). One representative of n = 11 independent experiments is shown. c Pten expression levels (transcript per million, TPM) from a 2 h-resolved RNA-seq differentiation time course in WT²⁸. d Western blot analysis for indicated proteins in WT and Pten KO in 2i/LIF, at N24 and N48. TUBULIN served as a loading control. e Quantification of pAKT (S473) levels by flow cytometry in indicated cells and conditions. Mean and standard deviation (SD) of mean fluorescence intensity (MFI) from n = 3 independent experiments (distinct shades of grey) are shown. p values result from paired, two-tailed t-tests. f Heatmap showing row-normalised expression values of indicated naïve and formative genes in indicated genotypes and conditions. g Scatter plots showing correlation between differentially expressed genes (DEGs, p adj. ≤ 0.05, |log2foldchange (log2FC)| ≤ 0.5) in Pten or Tsc2 KO in 2i and N24. Trend lines with 95% confidence intervals, and Pearson’s correlation coefficients (R) are shown. h Flow cytometry analysis of Rex1-GFP levels in indicated cell lines at N24, after indicated treatments. Rapamycin-treated WT cells are shown as dashed grey line. One representative of n = 5 independent experiments is shown. i Expression levels of indicated genes in indicated cell lines at N24 after indicated treatments measured by RNA-seq. p adj. values resulting from DESeq analysis are indicated in the plot. j Box plot showing expression of 377 naïve genes (naïve early and naïve late genes³³) in indicated KOs and conditions measured by RNA-seq. p values from two-tailed Wilcoxon signed rank tests are indicated. k Box plot showing the expression of 377 naïve genes in indicated KOs and conditions measured by RNA-seq. p value from two-tailed Wilcoxon signed rank test is indicated. All box plots in this paper show the 25th and 75th percentiles and median. Whiskers indicate minimum and maximum values.