Runx2 maintains pAkt levels in invasive MDA-MB-231 cells. A) The MDA-MB-231 cells stably expressing Runx2-RNAi or control were serum-deprived and stimulated with epidermal growth factor (EGF) (100 ng/ml). The whole cell lysates were analyzed for pAkt (Serine 473) and total Akt expression by Western blotting at indicated times. B) The average (± standard deviation) of normalized relative pAkt levels post EGF stimulation at indicated time points are shown (*P <0.05). C) The MDA-MB-231 cells were transfected with dsRNA to transiently knockdown Runx2 gene expression. The serum-deprived cells were stimulated with EGF and pAkt (Serine 473) level was analyzed. D) The Runx2 gene expression was conditionally suppressed by doxycycline in tTR-KRAB expressing MDA-MB-231 cells. The serum-deprived cells were stimulated with EGF and pAkt (Serine 473) level was analyzed. E) The stable Runx2 knockdown MDA-MB-231 cells were examined for pAkt (Serine 473) and pGSK-3β (Serine 9) expression by Western blotting. F) The pGSK-3β (Serine 9) expression was quantified from three blots and normalized expression level is shown. G and H) The stable (G) or conditional (H) Runx2 knockdown MDA-MB-231 cells were stimulated with EGF at indicated times. The whole cell lysates were examined for the expression of FOXO1 protein, while β-Actin was used as loading control. I) The WT-Runx2 (mouse) was re-expressed by Ad vectors at indicated multiplicity of infection (MOI) in Runx2-RNAi MDA-MB-231 cells. The whole cell lysates of serum-deprived and EGF treated cells were analyzed for pAkt (Serine 473) and the total Akt protein expression by Western blotting. The re-expression of mouse WT-Runx2 was also confirmed. J) The control or Runx2 knockdown MDA-MB-231 cells were utilized to re-express WT-Runx2 (mouse) or green fluorescent protein (GFP) via Ad vectors and were deprived of serum and glucose for 24 hours. The fixed cells were analyzed for sub-G1 population. A representative cell cycle profile is shown.