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. 2014 Sep 5;5(8):670–678. doi: 10.7150/jca.9688

Figure 4.

Figure 4

Akt2 signaling involves in EGF-activated cell viability. (A) Effects of EGF on Akt activation and status of Akt isoforms between OVCAR-3 and SKOV-3 ovarian cancer cells. Cells were treated with EGF (10 ng/mL) in a time-dependent manner (0, 5, 15, 30, 60 and 120 min). (B) Effect of siRNAs for Akt1 and Akt2 on EGF-activated Akt and Erk in OVCAR-3 cells. Cells were transiently transfected with siRNAs for Akt1 and Akt2 (final concentration 10 nmol/L) using the transfection reagent for 48 h followed by treatment with EGF (10 ng/mL) in a time-dependent manner (0, 5 and 15 min). (C) Effects of Akt1/2 siRNAs on EGF-induced cell viability in OVCAR-3 cells. Cells were transiently transfected with Akt1/2 siRNAs (final concentration 10 nmol/L) using the transfection reagent for 48 h followed by treatment for 48 h with EGF (10 ng/mL). (D) Effects of s Erk1/2 iRNAs on EGF-induced cell viability in OVCAR-3 cells. Cells were transiently transfected with Erk1/2 siRNAs (final concentration 10 nmol/L) for 48 h followed by treatment for 48 h with EGF (10 ng/mL). Blackened bars were EGF treated. * and ** indicate a significant increase (p≤0.05) between treatments by ANOVA and Tukey's pairwise comparisons. (E) The validation of Erk1 and Erk2 silencing. For western blot, whole cell lysates were prepared and immunoblots were carried out using antibodies specific for phosphorylated Akt (pAkt) and pErk, pan Akt and the three Akt isoforms (Akt1, Akt2 and Akt3). Pan Akt and β-actin were used as loading controls. Experiments were performed in duplicate and a representative result is shown. The cell viability assay was performed by using MTT, and values were normalized to untreated controls. Blackened bars were EGF treated. * and # indicate a significant increase and decrease (p≤0.05), respectively, by ANOVA and Tukey's pairwise comparisons. Experiments were performed in triplicate and all data are shown as mean ± SEM.