Figure 3

Rictor is essential for mTORC2 signaling upon PTEN mutation. (a–g) PTEN^mu cells were transfected with siRictor. After 24 h of transfection, cell lysates were prepared and subjected for western blot analysis. (a) Expression of Rictor was determined. (b, c) Level of mTORC-specific phosphorylations were shown in western blot with relative densitometry values. (d, e) mTORC2 activity was checked by studying Ser473 phosphorylation of AKT. Relative densitometry analysis of AKT ser473 showed. (f, g) Status of GSK3β Ser9 phosphorylation with relative densitometric analysis. (h) PTEN^wt cells were transfected with siPTEN or co-transfected with both siPTEN and siRictor consecutively. After 24 h of transfection, cell lysates were prepared and subjected for western blot analysis. Western blots showed that knockdown of Rictor from PTEN-depleted cell reverted mTORC2 signaling pathway. Single knockdown of PTEN increased phosphorylation of mTOR (Ser2481), AKT (Ser473) and GSK3β (Ser9). Depletion of both PTEN and Rictor decreased phosphorylation of mTOR (Ser2481), AKT (Ser473) and GSK3β (Ser9). (i) Status of p-AKT (Ser473), p-GSK3β (Ser9) and p-mTOR (Ser2481) with respect to total protein level in other PTEN^mu (T98G, U373MG) and PTEN^wt (LN18) GBM cell lines. β-Actin was served as loading control in all set of experiments. All the densitometric analysis were done by ImageJ analysis and normalized against the expression of β-actin and respective total protein level. Each value is the mean±s.d. of three independent experiments. *P<0.05, significant difference between two test groups.