Fig 2: Analysis of TRAIL receptor binding of TRAILR1 and TRAILR2 discriminating TRAIL mutants. (a) Hek293 cells, which express endogenous TRAILR2 and TRAILR4, were transiently transfected with expression constructs encoding the four types of cell-bound TRAIL receptors. Next day, equilibrium binding studies were performed at 37 °C with GpL-TNC-TRAIL, GpL-TNC-TRAILmutR1 and GpL-TNC-TRAILmutR2. Mock-transfected cells served to determine non-specific binding. (b) Equilibrium binding studies were performed with Jurkat cells, which only express TRAILR2 and the various GpL-TRAIL fusion proteins. Cells pretreated with 5 µg/ml TRAIL were used for determination of nonspecific binding. (c) Black 96-well ELISA plates were coated with protein G (0.5 µg/ml) and loaded with TRAILR1(ed)-Fc or TRAILR2(ed)-Fc (~1 µg/ml) or remained untreated for determination of non-specific binding. After removal of unbound molecules, the indicated concentrations of GpL-TNC-TRAIL, GpL-TNC-TRAILmutR1 and GpL-TNC-TRAILmutR2 were added for 1 h at 37 °C, and finally the well-associated luciferase activity was quantified. (d) HT29 cells were incubated (2 h, 37) with Fc-fusion proteins of the various TRAIL variants and were then immunoprecipitated by help of protein G agarose. Immunoprecipitates and cell lysates were analyzed by western blotting with respect to the presence of the indicated proteins. Lysates of untreated cells were supplemented with 10 ng of the corresponding Fc-fusion protein and served as a negative control

Fig 3: Alternol induces expression of DR5. (A) Caki-1 cells were treated with various doses of alternol (ALT) for 24 h. Total cellular extracts were then prepared and analyzed for DR4, DR5, DcR1, and DcR2 by western blot. (B) Caki-1 cells were treated with various doses of alternol (ALT) for 24 h. DR5, DR4, DcR1, and DcR2 mRNA levels were analyzed by qRT-PCR. (C) Cellular surface levels of DR5, DR4, DcR1, and DcR2 were analyzed by flow cytometry staining using PE-conjugated specific antibodies for each receptor. IgG isotype controls (gray histogram), DMSO (gray line), and alternol (red line) are presented. (D) Caki-1 cells were treated with various doses of alternol for 24 h or treated with 20 µM alternol for different times. Then, cellular lysates were analyzed with indicated antibodies. HCT116 wt and HCT116 p53-/- cells were treated with various doses of alternol for 24 h, and DR5 protein and mRNA levels were analyzed by western blot (E) and qRT-PCR (F), respectively. (G) Various types of carcinoma cells were treated with 20 µM alternol for 24 h, after which whole-cell extracts were analyzed by western blot assay. The same blots were stripped and re-incubated with actin antibody to confirm equal protein loading. Data are the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, compared with 0 µM ALT.