Fig. 2: Pten deletion maintains ESCs by regulating the PI3K-Gsk3β signaling axis.

a Phase-contrast images of WT and Pten^−/− ESCs cultured in medium including 2iL and in medium lacking PD0325901, CHIR99021, LIF, 2i, and 2iL, respectively. Scale bars, 100 µm. b, c Analysis of AP staining of WT and Pten^−/− ESC colonies cultured in medium including 2iL and in medium lacking PD0325901, CHIR99021, LIF, 2i, and 2iL, respectively. Without CHIR99021 and LIF, respectively, Pten^−/− ESC colonies displayed more than 50% domed morphology, but WT ESC colonies showed a large reduction in domed morphology. Error bars indicate mean ± SEM (n = 3), and 80 colonies were scored in each replicate. d Western blot analysis of WT and Pten^−/− ESCs cultured in medium without CHIR99021 showing the phosphorylation of Akt at T308 and S473 and the phosphorylation of Gsk3β at S9. e Western blot analysis of WT and Pten^−/− ESCs cultured in medium without LIF showing the phosphorylation of Akt at T308 and S473 and the phosphorylation of Stat3 at Y705. f Western blot analysis of Pten^−/− ESCs treated with PI3K inhibitors LY294002 and PX-866 showing the expression of the pluripotency markers and the phosphorylation of Akt at T308 and S473. g Protein levels of β-catenin in the cytoplasm/membrane and nucleus of WT and Pten^−/− ESCs. h Immunofluorescence staining for β-catenin in WT and Pten^−/− ESCs. DNA was stained with DAPI to indicate nuclei. Scale bars, 10 µm.