Figure 2

AKT1 regulated Notch-1 and PTEN expression through NFκB and had an anti-apoptotic role in gastric cancer cells. (a) Lysate from MKN-28 cells (parental), MKN-28 cells expressing lentiviral scramble shRNA (SCR shRNA), or lentiviral AKT1 shRNA were analyzed by anti-AKT1 blotting. The MKN-28 cells expressing lentiviral SCR shRNA or lentiviral AKT1 shRNA were treated with PS or 3 μM doxorubicin for 12 h, respectively. The protein and mRNA levels of AKT1, Notch1 and PTEN were detected by immunoblotting and real-time RT-PCR. Immunoblotting results of β-actin were used to show equal loading. Values are the mean±S.D. from three different experiments. ^★P<0.05, ^★★P<0.01 versus SCR shRNA group; *P<0.05, **P<0.01 versus SCR shRNA+Dox group. (b) Effect of doxorunicin and LY294002 (10 μM) on expression of AKT1, p-AKT (473), NICD, and PTEN. Immunoblotting results of β-actin were used to show equal loading. (c) The MKN-28 cells expressing lentiviral SCR shRNA or lentiviral AKT1 shRNA were treated with PS or 3 μM doxorubicin for 12 h, respectively. The DNA binding capability of NFκB was detected by EMSA analysis. (d) The MKN-28 cells and MKN-28 cells expressing lentiviral SCR shRNA or lentiviral AKT1 shRNA were treated with PS or 3 μM doxorubicin for 12 h, respectively. The DNA binding capability of CBF-1 on PTEN minimal promoter was detected by EMSA analysis. (e) MKN-28 cells’ preincubation with DMSO or 10 μM LY294002 and MKN-28 cells expressing lentiviral SCR shRNA or lentiviral AKT1 shRNA were treated with PS or 3 μM doxorubicin for 12 h, respectively. Cell apoptosis was determined by flow cytometry. ^★P<0.05, versus SCR shRNA group. ***P<0.01 versus SCR shRNA+Dox group. (f) Analysis of relative caspase-3 activity in cells. Normalized caspase-3 activity. All experiments were performed in triplicate. ^★P<0.05, versus SCR shRNA group. ***P<0.01 versus SCR shRNA+Dox group