Figure 6

Runx2 knockdown alters expression levels of mTORC-2 proteins. A) The MDA-MB-231 cells’ transient Runx2 suppression was analyzed for mTOR and Runx2 levels. B) A quantification of mTOR protein expression normalized to β-Actin is shown. C) The stable Runx2 knockdown MDA-MB-231 cells were serum-deprived, epidermal growth factor (EGF) stimulated and examined for pmTOR (Serine 2481) and mTOR (total) protein. D) The Runx2 knockdown cells were assayed for mTOR gene expression by RT-PCR (normalized to GAPDH). E) The Runx2 knockdown and Ad-GFP- or WT-Runx2-treated MDA-MB-231 cells were tested for Runx2 recruitment on mTOR promoter by ChIP assays. A schematic diagram of the mTOR promoter region is bars indicating potential Runx binding sites (black bars) and location of PCR primers (small arrows). F) and G) The mRNA (F) and protein (G) expression levels of Rictor were analyzed in control or Runx2 knockdown MDA-MB-231 cells by RT-PCR and Western blotting, respectively. H) The MDA-MB-231 cells stably over-expressing WT-Runx2 were assayed for Runx2 and Rictor gene expression by RT-PCR. I) The MDA-MB-231 cells with stable Rictor knockdown were stimulated with EGF and were analyzed for the expression of pAkt (Serine 473), Akt (total) and Rictor protein levels by Western blotting. J) The stably Runx2 knockdown MDA-MB-231 cells together with Rictor suppression were stimulated with EGF and analyzed for the expression of pAkt (Serine 473), Akt (total) and Rictor proteins by Western blotting. K) The glucose and serum-deprived (24 h) MDA-MB-231 cells with Runx2 knockdown (Runx2-RNAi) together with Rictor knockdown (Rictor -shRNA) were stained for Annexin V and AAD by flow cytometry. L) A quantification of positive cells with or without Rictor knockdown is shown. M) A schematic diagram depicting the function of Runx2 in regulating Akt via mTORC2 complex and its effect on survival of invasive cancer cells.