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. Author manuscript; available in PMC: 2009 Sep 21.
Published in final edited form as: Gynecol Oncol. 2008 Mar 7;109(2):240–249. doi: 10.1016/j.ygyno.2008.01.042

Figure 5. Expression of pro-survival markers and MAP-Kinase in SKOV-3 after 7Me-IEITC treatment; effect of MAP-Kinase inactivation on cell viability.

Figure 5

Panel A: Activation of MAP Kinases. SKOV-3 were treated with 4μM 7Me-IEITC for 1hr, 6hrs, or 18hrs. 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-beta-Actin antibody. As a size standard pre-stained Kaleidoscope (Biorad, Hercules, CA) marker was used.

Panel B: Effect of JNK and p38 inactivation on cell viability. SKOV-3 cells were pre-incubated with specific inhibitors (40μM) against JNK or p38 MAP-Kinase for 2hrs and treated with 7Me-IEITC (0, 2.5 or 5μM) in the continued presence of the inhibitors for additional 48hrs. As a positive control for the maximal potential effect of inhibition of p38 activity on of drug induced cytotoxicity, SKOV-3 cells were treated with a calcidiol derivative (+Ctr., 1μM B3CD) (Lange et al., in preparation). 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±SD) of a representative experiment in % cell viability of samples with untreated cells.

Panel C: Inactivation of survival signaling proteins and transcription factors. SKOV-3 were treated with 4μM 7Me-IEITC for 6hrs, 18hrs, or 36hrs. Analysis of the expression of proteins in the lysates of treated and untreated cells by PAGE and Western Blot analysis was carried out using primary antibodies against pro- and activated/phosphorylated PI-3K, STAT-3, IKKα, or NF-kB. As an internal standard for equal loading blots were probed with an anti-βActin antibody. As a size standard pre-stained Kaleidoscope (Biorad, Hercules, CA) marker was used.