Fig. 1

mTORC2 activation correlates with global DNA hypomethylation phenotypes in RTK-mutated GBM. A Immunohistochemical (IHC) staining of human GBM tissue (n = 20) with antibodies against mutant EGFR (EGFRvIII) and a DNA methylation marker (5-mC). EGFR amplification was assessed by FISH with probes for EGFR (7p11.2, Red) and CEP7 (7p11.1-q11.1, Green). Scale bar, 40 µm. B Cerebral and brainstem tissue with GBM tumors was harvested from rats infected with PDGFB-HA-IRES-EGFP retroviral vectors (n = 4). Immunohistochemistry was performed on paraffin-embedded tissue sections against a DNA methylation marker (5-mC) and an mTORC2 marker (p-Akt S473). Nec, necrosis. Scale bars, 50 µm (upper panels) and 20 µm (lower panels). C Immunofluorescent staining of 5-mC in U87-EGFRvIII cells transfected with shRNAs against control sequence (scramble) or Rictor (shRictor#1). Scale bar, 10 µm. D Dot blot analysis of 5-mC in U87-EGFRvIII cells transfected with shScramble versus shRictor#1 (upper panel), or overexpressed (OE) with GFP versus Rictor (lower panel). Total DNA for each sample was determined by methylene blue staining. E Detection of global DNA methylation (ELISA-based assay), represented by methylation of LINE-1 retrotransposable elements in U87-EGFRvIII cells transfected with shScramble or shRictor. OD, optical density; STD, standard