Figure 4

Expression of prosurvival markers and MAPKs in SKOV-3 following NB7M treatment; effect of MAPK inactivation on cell viability. (A) Activation of MAPKs. SKOV-3 cells were treated with 2 μ M of NB7M for 6, 18 or 36 h. Analysis of the expression of proteins in the lysates of treated and untreated cells by PAGE and western blot analysis was carried out as described (Material and Methods), using primary antibodies against pro- and activated/phosphorylated (P-) SAP/JNK, p38 and ERK1/2. As an internal standard for equal loading, the blots were probed with an anti-β-actin antibody. (B) Effect of p38 MAPK inactivation on cell viability. SKOV-3 cells were pre-incubated with specific inhibitors (40 μ M) against p38 MAPK for 2 h and treated with NB7M (0, 1 or 2 μ M) in the continued presence of the inhibitors for an additional 48 h. The MTS viability assay was carried out as described (Materials and Methods). Experiments were performed in triplicates; data are expressed as the mean of the triplicate determinations (X±s.d.) of a representative experiment in % cell viability of samples with untreated cells. (C) Effect of NB7M on DNA-pK and Axl. SKOV-3 cells were treated with 2 μ M of NB7M for 0, 6, 18 or 36 h. Analysis of the expression of proteins in the lysates of treated and untreated cells by PAGE/western blot analysis was carried out using primary antibodies against DNA-pK and Axl proteins. (D) Inactivation of survival signalling proteins and transcription factor proteins: SKOV-3 cells were treated with 2 μ M of NB7M for 6, 18 or 36 h. Analyses of the expression of proteins in the lysates of treated and untreated cells by PAGE/western blotting were carried out using primary antibodies against pro- and activated/phosphorylated PI-3K, STAT-3, IKKα or NF-κB.