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. 2013 Jul 4;4(7):e702. doi: 10.1038/cddis.2013.214

Figure 3.

Figure 3

Both cFLIP and XIAP provide TRAIL resistance in fibroblasts. (a) TRAIL induces pro-caspase-3 processing to the p20 fragment. hFb were treated with 200 ng/ml TRAIL-CL (3, 6, 12, 24 h) in the absence or presence of a pre-treatment with 10 μg/ml of CHX for 15 h (upper and lower panels, respectively) and 30 μg whole-cell lysate were analysed for pro-caspase-3 and PARP cleavage with western blotting. As a positive control, 30 μg of TRAIL-treated (50 ng/ml, 4 h) Colo205 cell lysate was used. (b) Knockdown of cFLIP increases TRAIL-mediated caspase-3 processing to the p20 form. Cells were transfected with an siRNA (50 nM) against cFLIP (cFLIP1) or GFP as transfection control. Twenty-four hours after transfection, cells were treated with 200 ng/ml TRAIL or TRAIL-CL for 24 h and whole-cell lysates were analysed for pro-caspase-3 processing and PARP cleavage with western blotting. As a positive control, 30 μg of TRAIL-treated (50 ng/ml, 4 h) Colo205 cell lysate was used. (c–e) Cells were transfected with siRNAs (50 nM) against cFLIP (cFLIP1) and/or XIAP or GFP as transfection control. (c) Lysates from transfected cells were analysed for cFLIP and XIAP expression. (d) Knockdown of cFLIP together with XIAP sensitises hFb to TRAIL. Twenty-four hours after transfection, cells were treated with 200 ng/ml TRAIL-CL for 24 h and induction of apoptosis was measured with Annexin V analysis. *P<0.05. (e) cFLIP+XIAP knockdown leads to processing of pro-caspase-3 to its p17 form in response to TRAIL. Twenty-four hours after transfection, cells were treated with 200 ng/ml TRAIL or TRAIL-CL for 24 h and cell lysates were analysed by western blotting for pro-caspase-3 and PARP cleavage. As a positive control, 30 μg of TRAIL-treated (50 ng/ml, 4 h) Colo205 cell lysate was used. (f) Knockdown of cFLIP+XIAP specifically sensitises to TRAIL-induced apoptosis and not to other apoptotic stimuli. Cells were transfected with the indicated siRNAs (50 nM). Twenty-four hours after transfection, cells were treated with 100 μM etoposide or 10 μM doxorubicin for 24 h and induction of apoptosis was measured with Annexin V assay. All graphs show mean values±S.E.M.