Fig. 3

Disruption of Ras-mSIN1 interaction leads to the inhibition of mTORC2 signaling. a Pre-treatment (24 h prior to Pyrogallol) of MDA-MB-231 and DU 145 cells with peptides synthesized on the basis of the amino acid sequence of the RBD within mSIN1 revealed diminished mTORC2 signaling compared to that in cells treated with Pyrogallol (20 μM) alone. Maximum inhibition was observed with peptide designated P4. b Co-immunoprecipitation studies carried out in MDA-MB-231 cells pre-treated with P4 and Scr peptide for 24 h followed by Pyrogallol stimulation revealed diminished interaction between Ras and mSIN1 in presence of P4 compared to that in Pyrogallol treated or cells treated with scrambled peptide. c In situ proximity ligation assay (PLA) revealed that direct physical interaction between Ras and mSIN1 was hampered in cells that were pre-treated with P4. d Western blot analysis of cytosolic fraction, membrane fraction and whole cell lysate revealed increased levels of Ras and mSIN1 in membrane fraction with a corresponding decline in the cytosolic fraction following Pyragallol treatment. Pre-treatment with Scrambled peptide did not alter cellular redistribution of either Ras or mSIN1. Pre-treatment with P4 diminished mSIN1 localization to membrane fraction without altering Ras levels in the membrane fraction. The mTOR followed a pattern similar to mSIN1. e Constitutive Ras activation with GTP-γ-S treatment for 12 mins in cell lysates of un-treated MDA-MB-231 and DU 145 cells, and ensuing increased interaction of Ras with mSIN1 has hindered cells that were treated with P4 (24 h prior to harvest). f Constitutive Ras activation with GTP-γ-S treatment for 12 mins in cell lysates of un-treated MDA-MB-231 and DU 145 cells and ensuing mTORC2 signaling was hindered in cells that were treated with P4 (24 h prior to harvest). g The Immunoblot analysis of in vitro kinase assay using mTORC2 immunopurified from MDA-MB-231 cells treated with P4 either alone or in combination with Pyrogallol shows diminished kinase activity as compared to reactions containing mTORC2 immunopurified from Pyrogallol treated or untreated cells. h MDA-MB-231 cells treated with P4 (50 μg/ml for 24 h) prior to Pyrogallol exposition (20 μM for the further 12 and 24 h) exhibited diminished cell migration compared to those that were pre-treated with scrambled Peptide. Scale bars: 100 μm. i MDA- MB-231 cells treated with P4 (50 μg/ml for 24 h) prior to Pyrogallol exposition (20 μM for the further 24 h) exhibited diminished invasion through Matrigel. Cells were stained with DAPI. n = 3 biological replicates per group. All data are representative of three independent experiments. ns (not significant). *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001