Figure 2.

TRAIL can activate the JNK pathway via both DR4 and DR5 in colon cancer cell lines. (A) Western blot analysis of total JNK and p-JNK in Colo205, HCT15 and HCA7 cell lysates following treatment with 20 ng ml⁻¹ rhTRAIL for Colo205 cells and 50 ng ml⁻¹ for HCT15 and HCA7 for the times indicated. Total JNK levels and actin were also detected as loading controls. (B) Western blot analysis of total c-Jun and p-c-Jun levels in Colo205, HCT15 and HCA7 cell lysates following rhTRAIL treatment as above. (C) Cell surface expression of DR4 and DR5 in Colo205 and HCT15 cells measured by immunostaining followed by flow cytometry. Each histogram shows an overlay of a negative control (2° AB), DR4 and DR5 receptors. (D) Apoptosis-inducing potential of DR4 and DR5 in Colo205 and HCT15 cells. Cells were treated with cross-linked agonistic DR4 or DR5 antibodies (5 nM for Colo205 and 10 nM for HCT15) or rhTRAIL (20 and 50 ng ml⁻¹ for Colo205 and HCT15) for 3 h in Colo205 cells and for 5 h in HCT15 cells. Apoptosis induction was measured with Annexin V. The graphs show averaged percentage of apoptotic cells±s.e.m. of three independent experiments. (E) Western blot analysis showing activation of JNK via DR4 and DR5 in Colo205 and HCT15 cells. Cells were treated with agonistic DR4 and DR5 antibodies (5 nM for Colo205 cells and 10 nM for HCT15 cells) for the times indicated. JNK phosphorylation was detected in whole cell lysates. The images shown are representatives of three independent experiments. Actin was detected as a loading control.