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. 2015 May 26;10(5):e0126526. doi: 10.1371/journal.pone.0126526

Fig 1. Ezrin inhibits TRAIL-induced apoptosis downstream of the DISC.

Fig 1

(A) HCT116 or (B) SW480 cells, expressing or not VSV-ezrin were treated for 6 hours with Fas ligand (100 ng/ml) or His-TRAIL (500 ng/ml) or 16 hours with 1μM staurosporin (STS). Apoptosis was quantified by Hoechst staining. Data represent the mean ± SD of at least three different experiments. (*P<0.05; **P<0.01 respective to control cells). Ezrin ectopic expression levels were analyzed by immunoblotting using an anti-VSV antibody in control or ezrin WT-expressing HCT116 and SW480 cells. HSC70 was used as a loading control. (C) HCT116 or (D) SW480 cells, were transfected ezrin or scramble (Scr) siRNAs. 72 h after transfection cells were stimulated for 6 hours with Fas ligand (100 ng/ml) or His-TRAIL (500 ng/ml) and apoptosis was quantified after staining with APO2.7 antibody by flow cytometry. Data represent the mean ± SD of at least three different experiments. (*P<0.05; **P<0.01 respective to control cells). Ezrin expression levels were analyzed by immunoblotting. Actin was used as a loading control. (E) Analysis of TRAIL DISC formation. HCT116 and SW480 cells were stimulated or not with 5 μg/ml Flag-TRAIL cross-linked with 10 μg/ml anti-Flag (M2) antibody. Cells were lysed, and the DISC was immunoprecipitated and analyzed by western blot. One of three independent experiments is shown. (F) HCT116 cells were stimulated or not with 5 μg/ml His-TRAIL for 20 and 60 minutes. After cell lysis, GAPDH antibody was added to the cell lysates and immunoprecipitates were analyzed by western blot.