Fig 2: Taurine augments PTEN activity inhibited mTOR signaling and activated autophagy. (A,B) Confocal images of LC3B puncta (red dot) in MAC-T cells incubated with 2 µmol·L-1 VOTH (VO-OHpic trihydrate) for 24 h before treatment with 70 mmol·L-1 taurine for 4 h. (C) PIP2 and PIP3 concentrations were determined by ELISA in the same cells. (D–G) Representative immunoblots of total protein from MAC-T cells. Quantification of the ratio between total and phosphorylated (p–) proteins, as determined by densitometric scanning of immunoblots and normalization to GAPDH (n = 3). Data are presented as mean ± SEM. *P < 0.05 (significantly different) between the indicated groups.

Fig 3: Lipid phosphatase activity of PTEN I101T mutant. Empty vector control (C), PTEN WT and I101T expression plasmids were transfected in either PC3 (A) or U251 (B) cell lines. The levels of p-Akt S473 (p-Akt) and total Akt (c-Akt) were examined by Western blotting analysis. Specific p-Akt levels were calculated from six and three individual experiments for PC3 and U251, respectively. Bars, SD. *P < 0.05; **P < 0.01; ns, not significant; Mann–Whitney test. (C) PC3 transfected with indicated plasmids were serum-starved for overnight and stimulated with 10% FBS for 10 min. The levels of p-Akt T308, p-Akt S473 and total Akt (c-Akt) were examined by Western blotting analysis. Specific p-Akt levels were calculated from three individual experiments. ns, not significant; Mann–Whitney test. (D) Indicated plasmids were transfected in 293T cells. PTEN was immunoprecipitated by an anti-AU5 antibody and probed with an anti PTEN antibody (lower panel). PTEN protein levels were normalized and lipid phosphatase assay was carried out with PIP2+PIP3 as substrates with released phosphate measured by a Malachite Green Assay kit (Echelon). Lipid phosphatase activities from seven independent experiments were quantified and expressed as pmol ng-1min-1. Bars, SD. **P < 0.01, ***P < 0.001; Mann–Whitney test.

Fig 4: Pten-inhibitor SF1670 plays a similar role to CHIR99021 in maintaining the pluripotency of ESCs.a Phase-contrast images of DMSO-treated and SF1670-treated WT ESCs cultured in medium without CHIR99021. Scale bars, 100 µm. b Q-PCR analysis of pluripotency marker (Esrrb, Zfp42, and Fgf4) expression in DMSO-treated and SF1670-treated ESCs cultured in medium without CHIR99021 from day 0 to day 7. Error bars indicate mean ± SD (n = 3). c Western blot analysis of WT ESCs treated with DMSO and SF1670 in medium without CHIR99021 showing the phosphorylation of Akt at T308 and S473 and the phosphorylation of Gsk3ß at S9. d Nude mice were injected with DMSO-treated and SF1670-treated WT ESCs on the left and right sides, respectively. Teratomas were generated at the contralateral positions aligned at the same positions in each treatment. e Weights (g) of teratomas from DMSO-treated and SF1670-treated ESCs. f DMSO-treated and SF1670-treated ESCs were injected into immunodeficient mice and produced teratocarcinomas containing tissues representative of the three germ layers (mesoderm, ectoderm, and endoderm).

Fig 5: siRNA-mediated knock-down of PTEN or inhibition of PTEN with bpV(pic) promotes NSC differentiation. (a) Immunoblot analysis of PTEN, ß-III-tubulin and ß-actin levels in NSCs after incubation with NIM for 0, 1, 3, 5, 7, and 14 days; n = 3 independent experiments. (b) Quantification of the levels of PTEN expression from day 0 to 14. n = 3 independent experiments. (c) Immunoblot analysis of PTEN, ß-III-tubulin, Nestin and ß-actin expression in NSCs following transfection with PTEN siRNA or vehicle, stimulated with NIM for 3 days, n = 3 independent experiments; (d) the expression levels of PTEN, ß-III-tubulin, GFAP, GLAC and Nestin in NSCs as measured by QPCR. NSCs were transfected with PTEN siRNA or vehicle and stimulated with NIM for 3 days, n = 3 independent experiments; (e) immunoblot analysis of p-PTEN, ß-III-tubulin and ß-actin in NSCs treated with different concentrations of bpV(pic) (0, 1, 5, 10, 20 or 50 ng ml-1) and NIM for 3 days, n = 3 independent experiments; (f) immunoblot analysis of p-PTEN, ß-III-tubulin and ß-actin in NSCs treated with vehicle or 20 ng ml-1 bpV(pic) and NIM for 1, 3 and 7 days, n = 3 independent experiments; (g) representative images of the immunofluorescence staining analysis of ß-III-tubulin in NSCs treated with vehicle or 20 ng ml-1 bpV(pic) and NIM for 1, 3 days, scale bar = 50 µm; (h) quantification of the average axonal length, n = 5, *p < 0.05, **p < 0.01, all data are shown as mean ± SD.