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Figure 2 | BMC Cancer

Figure 2

From: MUC16 mucin (CA125) attenuates TRAIL-induced apoptosis by decreasing TRAIL receptor R2 expression and increasing c-FLIP expression

Figure 2

MUC16 knockdown enhances TRAIL-induced activation of caspase-8 and apoptosis. (A) Cells were incubated for 48 h with increasing concentrations of TRAIL. Cell viability was measured by the XTT assay. All assays were performed in triplicate and repeated three times. Data are expressed as the mean of pooled data from all three experiments: bars, ± SEM. (B) Cells were incubated in the presence or absence of TRAIL (100 ng/ml) for 24 h, and morphological changes were assessed by phase contrast microscopy. Representative fields from one of two independent experiments are shown. (C) Cells were treated with anti-TRAIL-R2 agonist for 48 h. Cell viability was measured by the XTT assay. All assays were performed in triplicate and repeated three times. Data are expressed as the mean of pooled data from all three experiments: bars, ± SEM. (D) Cells were treated with TRAIL (100 ng/ml) for 24 h and stained with PI, and the subG0/G1 DNA content was analyzed by flow cytometry. Results from two independent experiments are shown. (E) Cells were treated with TRAIL (100 ng/ml) for up to 8 h and cell lysates were analyzed by immunoblots. (F) Cells were incubated with TRAIL for the indicated times and cell lysates were immunoblotted with anti-Bid antibody. The anti-Bid antibody used does not detect the truncated form of Bid. (G) Cells were cultured for 24 h without TRAIL and the mitochondrial membrane integrity was assessed using MitoLight™. In treated cells, fresh culture medium containing 200 ng/ml of TRAIL was added for 45 min prior to MitoLight™ staining. Cells containing red-labeled mitochondria are scored as viable cells. (H) OVCAR3 and isogenic TRAIL resistant R350 cells were treated with TRAIL (100 ng/ml) for 48 h and cell viability was assessed by XTT. Immunoblot showing expression of MUC16 in the upper panel.

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