Figure 4

Runx2 specifically maintains pAkt in invasive mammary epithelial cells. A) The basal levels of pAkt (Serine 473) expression was examined in MDA-MB-231, SUM-159 and SUM-159-PT cells cultured in regular growth medium by Western blotting. B) The Runx2 protein expression was stably suppressed in SUM-159 cells by lentivirus-mediated Runx2 shRNA delivery. The Runx2 expression levels were analyzed by Western blotting while β-Actin was used as the loading control. C) The SUM-159 cells expressing Runx2-RNAi or control were serum-deprived and stimulated with 100 ng/ml of EGF in the presence of 20 μM LY294002. The whole cell lysates were analyzed for pAkt (Serine 473) and total Akt expression by Western blotting at 10 minutes post epidermal growth factor (EGF) stimulation. A quantification of normalized relative pAkt levels is given below respective lane. D) The SUM-159-PT cells were transfected with dsRNA targeting a different Runx2 mRNA sequence to transiently suppress Runx2 expression. E) The serum-deprived, EGF stimulated SUM-159-PT cells were analyzed for pAkt (Serine 473) and Akt levels by Western blotting. A quantification of normalized pAkt level is shown below the blot. F) The MDA-MB-231 cells expressing Runx2-RNAi or control were serum-deprived and stimulated with 100 ng/ml of EGF in the presence of 20 μM LY294002. The whole cell lysates were analyzed for pAkt (Serine 473) and total Akt expression by Western blotting at 10 minutes and 1 hour post EGF stimulation.