Fig. 6: Role of PTEN in the regulation of stemness property in NS-induced cells.

a Representative phase-contrast images of PTEN^wt LN229 after 5 days of NS induction (iLN229) showing no sphere-like morphology. b Flow cytometric analysis showed no significant shifting of iLN229 toward smaller-sized R2 population after day 2 (D2) and day 5 (D5). c Western blot analysis showed a lower level of β-catenin and Gli1 in iLN229 (PTEN^wt) compared to iU87MG (PTEN^mu). d LN229 (5 × 10⁵) cells were transfected with PTEN shRNA using Lipofectamine LTX and Plus reagents followed by 5 days of nutritional stress. Representative immunoblots showing an increased level of β-catenin, Gli1, and Gli2 in PTEN-knockdown iLN229. e PTEN-knockdown iLN229 showed higher GSK3β phosphorylation at the Ser9 position, leading to lower GSK3β activity compared to mock-transfected iLN229, which is mediated by higher AKT phosphorylation at Ser473. f PTEN-knockdown iLN229 showed higher expression of GSCs markers compared to mock-transfected iLN229 as assessed by flow cytometry. g Representative phase-contrast images showing larger neurosphere formation in PTEN-knockdown iLN229 compared to mock-transfected iLN229. h Quantitative representation of number and size of neurospheres in PTEN-knockdown iLN229. i Schematic representation of nutritional stress-mediated emergence of GSCs by modulating Wnt and Hedgehog signaling and involvement of PTEN. Results represent mean ± SD from three independent experiments