Figure 5.
mTORC2 regulates stemness of GBM stem-like cells. (a) Representative immunoblot and coimmunoprecipitation demonstrated higher mTORC2 activity and formation as evidenced by increased mTOR Ser2481 phosphorylation and association with Rictor in CD133-positive stem-like cells derived from U87MG. APC-conjugated CD133 antibody was from Mylteni Biotech. (b) Immunoblot analysis showing enhanced mTOR Ser2481 and Gli2FL in GBM stem-like cells compared with normal human glial stem cells. (c) Rictor was overexpressed in LN229 cells. Simultaneously, Gli2 was knocked down in Rictor-overexpressed LN229 cells and cultured in stem cell medium. Rictor-overexpressed cells showed enhanced neurosphere formation but were unable to form large colonies when Gli2 was knocked down. (d) Both the number of CD133-positive cells and their expression were higher in Rictor-overexpressed LN229 cells as revealed by flow cytometric analysis. (e) Representative western blots showed increased levels of Oct4, Sox2, Nanog, Integrin α6 and Nestin in Rictor-overexpressed LN229 cells. Nestin antibody was from Biolegend. Statistical significance compared with the control is indicated by *P<0.05