Fig. 2

Superoxide anion stimulated the activation of mTORC2 is a Ras-mediated phenomenon. a Immunofluorescence micrograph depicting alteration in the cellular distribution of Ras 24 h following Pyrogallol (20 μM) treatment. Scale bars, 10 μm. b Subcellular fractionation followed by Western blot analysis depicting the increased distribution of Ras from cytosolic fraction to membrane fraction 24 h post-pyrogallol (20 μM) treatment. c MDA-MB-231 and DU 145 cells treated with Ras inhibitors FTI (1 μM) and GGTI (38 nM) for 24 h prior to Pyrogallol treatment of further 24 h exhibited impaired mTORC2 signaling. d An in vitro reconstitution assay wherein constitutive Ras activation was achieved by incubating MDA-MB-231 and DU145 cell lysates with GTP-γ-S (200 μM) for 12 mins revealed heightened mTORC2 signaling in the presence of active Ras. e MDA-MB-231 and DU 145 cells expressing wild-type H-Ras exhibited heightened mTORC2 signaling following Pyrogallol (20 μM) treatment. Comparable mTORC2 activation was observed in MDA-MB-231 and DU 145 cells, expressing constitutively active, mutant H-Ras (G12 V). f Co-immunoprecipitation followed by immunoblot analysis showing an increased association of Ras with mSIN1 in MDA-MB-231 and DU145 cells treated with Pyrogallol (20 μM) for 24 h. g In situ proximity, ligation assay revealed direct physical interaction between Ras and mSIN1 which further increased upon exposition to Pyrogallol for 24 h. h MDA-MB-231 and DU 145 cells pre-treated with Ras inhibitors viz. FTI/GGTI (1 μM/38 nM) for 24 h exhibited diminished interaction between mSIN1 and Ras despite Pyrogallol exposition. i In an in vitro reconstitution assay wherein constitutive Ras activation was achieved by incubating MDA-MB-231 and DU 145 cell lysates with GTP-γ-S (200 μM) for 12 mins. The co-immunoprecipitation study revealed increased interaction between mSIN1 and active Ras. j HA pull-down assay in ectopically HA-Ras expressing MDA-MB-231 and DU 145 cells exhibited increased co-immunoprecipitation of mSIN1 with HA-Ras, following exposition to Pyrogallol. All data are representative of three independent experiments. ***P ≤ 0.001