Fig. 2

RPPA analysis of mTORC1 and mTORC2 activity. GBM3-Luc, ADF and U373MG cells were incubated in the absence or in the presence of SI113 and then processed for RPPA and Western blot (left and right panels, respectively). SI113 was employed at the IC30 and IC50 concentrations specific for each cell line. Unless otherwise specified all RPPA plots refer to results obtained at 8 h of exposure to SI113. When significant, statistical comparisons (Wilcoxon signed-rank test, FDR-adjusted p values) are reported on each individual plot. Statistical significance coding is described in the Materials and Methods section of the manuscript. a. mTORC1. RPPA plots represent the trend of mTOR pS2448 and S6 pS235–36 phosphorylation as readouts of mTORC1 activation status. mTOR pS2448 and S6 pS235–36 normalization was performed by GAPDH quantification. b. mTORC2. Representative RPPA plots of SGK1 pS422 (2 h time point shown) and AKT pS473 show the trend of mTORC2 activity upon treatment with SI113. SGK1 pS422 was normalized against the GAPDH determination previously used for mTOR pS2448, while AKT pS473 in U373MG cells shares the loading control (GAPDH) with S6 pS235–36. c. AKT and SGK1 activity. RPPA plots represent the phosphorylation trend of MDM2 and NDRG1, which are targets of the AKT/SGK1 activity, under the effect of SI113. Nucleolin content was used for MDM2 pS166 normalization while GAPDH, the same as the one reported for SGK1 pS422 normalization in panel B, was used for NDRG1 pS330 normalization. d. Apoptosis. RPPA plots display the trend of cleaved PARP (D214) after SI113 treatment. GAPDH determination used for PARP D214 normalization in GBM3-Luc and ADF cells was done on the same filter used for AKT pS473. e. Autophagy. Plots of ACACA pS79 and AMPK-α pT172 RPPA levels are shown here to represent the trend of the autophagic process under the effect of SI113. ACACA pS79 in U373MG cells share the same GAPDH normalization used for AKT pS473 and S6 pS235–236. AMPK-α pT172 in GBM3-Luc and ADF cells share the same GAPDH normalization used for S6 pS235–236.kDa = apparent molecular mass