Fig. 4

a MDA-MB-231 and DU 145 cells were transfected with control vector, wild-type FLAG-mSIN1, and mutant FLAG-mSIN1-containing expression vectors followed by Pyrogallol stimulation for 24 h. Compared to MDA-MB-231 and DU 145 cells overexpressing Flag-tagged, wild-type mSIN1, the ones expressing Flag-tagged, mutant mSIN1 (Tyr-323 and Leu-325 replaced with Ala) exhibited diminished Ras-mSIN1 interaction. b Compared to cells overexpressing Flag-tagged- wild-type mSIN1, the ones expressing Flag-tagged-mutant mSIN1 (Tyr-323 and Leu-325 replaced with Ala) exhibited diminished mTORC2 signaling. c PTEN-deficient cell line PC-3 exhibited elevated activation of mTORC2 compared to DU 145 cells as shown by Immunoblot analysis. d Western blot analysis of lysates from PTEN-deficient LipPD1, LipPD2, LipPD3 lipoma cells exhibited diminished activation of mTORC2 signaling cascade as compared to PTEN wildtype-preadipocytes. e Western blot analysis of lysates from PC-3 cells treated with Rapamycin for various time points shows heightened mTORC2 signaling at 3 h and gradually decreases. f LipPD1 cells treated with Rapamycin at various time points exhibited heightened mTORC2 activation with most prominent effects at 3 h post-treatment. g Immunoblot analysis of cell lysates of PC-3 cells, pre-treated with P4 for 24 h followed by rapamycin for 3 h as indicated. Pre-treatment with P4 show decreased activation of mTORC2, alone and in combination with Rapamycin. All data are representative of three independent experiments. ns, not significant. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001