Fig. 4

MHY4571 induces apoptosis through, at least in part, the PKA/CREB axis in human squamous carcinoma lung cancer cells. A NCI-H1703 cells and HCC95 cells were treated with indicated concentrations of MHY4571 for 24 h, and western blot analyses were conducted to investigate the expression of P-CREB and CREB. B, C NCI-H1703 cells and HCC95 cells were treated with MHY4571 at 0, 5, and 7.5 µM for 24 h, and then the mRNA expression levels of E2F1, E2F2, and E2F8 were analyzed by quantitative RT-PCR and western blot analyses. β-actin was used as an internal control. Significance was determined using student's t-test (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. vehicle-treated cells). D, E NCI-H1703 cells and HCC95 cells were transfected with siPKA, siCREB, and scramble siRNA (negative control) for 48 h, and then the expression levels of P-PKA, PKA, P-CREB, and CREB proteins were analyzed by western blot. β-actin was used as a loading control. Significance was determined using student’s t-test (*P < 0.05 and ***P < 0.001 vs. scramble siRNA transfected cells). F NCI-H1703 cells and HCC95 cells were transfected with siPKA, siCREB, and scramble siRNA (NC) for 24, 48, and 72 h, and then MTT assay was performed to confirm cell viability. Results are expressed as the means ± SD of three individual experiments. Significance was determined using student’s t-test test (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. scramble siRNA transfected cells (NC))