FIGURE 4.

Induction of CHOP is required for cryptotanshinone-induced DR5 expression. A (top), Western blotting of A375 lysates from cells treated with 20 μm CT for indicated time periods; bottom, RT-PCR analysis of CHOP. B, A375 cells were pretreated with 1.5 μg/ml cycloheximide (CHX) or 2 μg/ml actinomycin D (ACT) for 30 min before incubation with 20 μm CT for 12 h. The whole-cell extracts were subjected to Western blot analysis using the anti-DR5 and anti-CHOP antibodies. C, A375 cells were transfected with pGL3-CHOP-643/+36 and then treated with 20 μm CT. After 12 h, cells were lysed and assayed for luciferase activity. *, p < 0.05 compared with vehicle-treated cells. D, luciferase assay with reporter pDR5-310/+293 was performed as described above. E, cells were transfected with pDR5-310 or pDR5-310ΔCHOP and then treated with 20 μm CT for 12 h and then assayed for luciferase activity. *, p < 0.05 compared with CT-treated pDR5-310-transfected cells. F (top), cells were transfected with siRNAs and cell extracts were prepared for Western blot analysis of DR5 and CHOP; bottom, after transfection with siRNA for 24 h, A375 cells were pretreated with 20 μm CT for 12 h, washed with PBS, and then treated with TRAIL (25 ng/ml) for an additional 24 h. Cell viability was assessed by the MTT assay. *, p < 0.05.