MUC16CTD is sufficient to attenuate TRAIL-induced apoptosis and caspase-8 activation in SKOV3 cells. (A) Lysates from SKOV3-EV or SKOV3-MUC16CTD were immunoprecipitated with anti-His or anti-c-myc-789 antibodies and subjected to immunoblotting with anti-c-myc 9E10 antibody. (B) SKOV3 cells expressing MUC16CTD or the empty vector (EV) were treated for 48 h with increasing concentrations of TRAIL. Cell viability was measured by the XTT assay and the percentage of cell viability was defined as the relative absorbance of treated versus untreated cells. All assays were performed in triplicate and repeated three times, and data are expressed as the mean of triplicate samples: bars, ± SEM. (C) Morphological analysis under phase contrast microscopy of SKOV3 cells treated with TRAIL (500 ng/ml) was also performed. Representative fields of one of three independent experiments are shown. (D) SKOV3-EV or SKOV3-MUC16CTD cells were incubated in the presence or absence of 500 ng/ml TRAIL and apoptosis was assessed by flow cytometry by analyzing the subG0/G1 DNA content. The percentage of apoptotic cells is indicated. Results from two independent experiments are shown. (E) SKOV3-EV and SKOV3-MUC16CTD cells were treated with TRAIL (500 ng/ml) for up to 6 h and cell lysates were analyzed by immunoblots using anti-caspase-8, anti-caspase-3 and anti-caspase-9 antibodies. The pro-caspase forms and their cleavage products are indicated by the arrows to the left of the panels. (F) Fluorescence microscopic analysis of the mitochondrial membrane permeability in SKOV3 cells. SKOV3-EV and SKOV3-MUC16CTD cells were cultured for 24 h without TRAIL and the mitochondrial membrane integrity was assessed using the MitoLight™. In treated cells, fresh culture medium containing 500 ng/ml of TRAIL was added for 60 min prior to MitoLight™ staining.