Figure 4
mTORC2 regulates the hedgehog pathway via GSK3β-dependent mechanism. (a) Rictor was either silenced in U87MG cells or overexpressed in LN229 cells. Representative immunoblots demonstrated increased or decreased GSK3β Ser9 phosphorylation upon Rictor-knocked-down or overexpression conditions. (b) Either Rictor alone or both Rictor and GSK3β were knocked down in U87MG cells. In parallel, GSK3β were also knocked down in LN229 cells before and after Rictor overexpression. Representative immunoblots showed enhanced Gli1, Gli2FL and Ptch1 protein levels in Rictor-knocked-down U87MG cells. However, the levels of all three proteins were reduced when both Rictor and GSK3β were knocked down. In contrast, GSK3β-knocked-down LN229 exhibited increased level of all these proteins, whereas Rictor overexpression in GSK3β-knocked-down LN229 cells showed more enhanced protein levels. (c) LN229 cells were treated either with MG132 (inhibitor of ubiquitination) or GSK3β inhibitor SB17237 (Calbiochem USA) separately for 24 h. Representative immunoblot showed restoration of Gli2FL protein in LN229 cells in a dose-dependent manner. (d) Gli2 was overexpressed in LN229 cells using vector encoding full-length Gli2. Real-time PCR analysis exhibited enhanced Gli1, Gli2, Ptch1, cyclin D1, cyclin D2, slug, snail and VEGF mRNA expression in Gli2-overexpressed LN229 cells relative to that of untransfected cells. Values are normalized against 18S rRNA expression (n=3 experiments). (e) Representative immunoblots showed enhancement of hedgehog pathway proteins and its downstream molecules upon Gli2 overexpression in LN229 cells. (f) Representative western blot showed lower nuclear accumulation of Gli1, Gli2FL in LN229 cells compared with U87MG cells. HDAC3 and β-actin served as a loading control. (g) Representative western blot showed higher association of Gli2FL and Sufu revealed by coimmunoprecipitation in LN229 cells. (h) Coimmunoprecipitation illustrated higher association of Smo with Rictor in U87MG cells and Rictor with Sufu in LN229. Statistical significance compared with the control is indicated by **P<0.01 and ***P<0.001
