Figure 6.

mTOR inhibition leads to SRC activation. (A) U87MG cells were cultured in complete DMEM (CTR) or aminoacid- and serum- free medium (EBSS) or in DMEM in the presence of 250 nM Torin1 or 100 nM AZD8055 for 18 h. Western blot analysis was performed by using specific antibodies for the total (SRC) and the phosphorylated (Y416) form of SRC (P-Src). β-ACTIN was used as loading control. (B) U87MG cells were cultured in DMEM in the presence of Torin1 pre-incubating or not with 20 µM PP2 inhibitor for the indicated time points. Western blot analysis was performed by using specific antibodies for the total (SRC) and the phosphorylated (Y416) form of SRC (P-Src). β-ACTIN was used as loading control. The graphs represent the mean ± SD of three different experiments. Statistical significance: ** p < 0.01 Student t-test, *** p ≤ 0.001 Student t-test (C) Immunocytochemistry and confocal analysis for EGFR localisation (red) were performed in U87MG, upon 6 h and 24 h of Torin1 and PP2 treatments, as indicated. Hoechst 33342 was used to stain nuclei (blue). Scale bar, 30 μM. (D) U87MG cells were cultured in DMEM in the presence of Torin1 pre-incubating or not with 20 µM PP2 inhibitor for the indicated time points. Western blot analysis was performed by using specific antibodies for the total (ERK1/2) and the phosphorylated form of ERK1/2 (P-ERK1/2). HSP90 was used as loading control. The graph represents the mean ± SD of three different experiments. Statistical significance: * p < 0.05 Student t-test, ** p < 0.01 Student t-test.