Figure 1.

Antiproliferative effects by BMS-345541. (a) Cell cycle analysis of melanoma cell lines A-375, Mel-HO, MeWo and Mel-2a. Cells treated with BMS-345541 (2 μM, open graphs) were compared with dimethylsulfoxide (DMSO)-treated controls (filled graphs). (b) Apoptosis induction (% of sub-G1 cells) and (c) cytotoxicity (LDH release) is shown in response to increasing concentrations of BMS-345541 (2–10 μM). (a–c) At least two independent experiments with triplicates revealed comparable results. (d) Total protein levels of I-κBα and nuclear levels of p65 were determined by western blotting in A-375 cells treated with TNF-α (10 ng/ml and BMS-345541 (2 μM). Total proteins were extracted at 0.25 h and nuclear extracts at 1 h of treatment. (e) Proteins and nuclear extracts were isolated at the same times after treatment with TRAIL and BMS-345541. (f) Relative p65 DNA-binding capacity in nuclear extracts of A-375, treated for 1 h with TNF-α (20 ng/ml), TRAIL (10 ng/ml) and BMS-345541 (2 μM), was quantified by enzyme-linked immunosorbent assay (ELISA). Means and S.D.'s were calculated from three independent experiments, and statistical significance is indicated (*P<0.05). (g) Nuclear extracts were analyzed by western blotting for p65 in A-375 and A-375-TS at 16 h of treatment with TRAIL (20 ng/ml) and BMS-345541 (A-375, 2 μM; A-375-TS, 5 μM). (d, e, g) At least two independent experiments revealed largely comparable results; equal loading was proven by GAPDH, β-actin and Ponceau staining