Figure 7

Analysis of biochemical changes in human malignant cell lines treated with poly(I:C) combined to RMT 5265. A) Cell samples representative of five different cell lines were subjected to the following treatments: control compound or RMT 5265 (100 nM for 48 h) with or without poly(I:C) (500 ng/ml; last 16 h). Caspase 3/7 (middle panel) and caspase 8 (upper panel) activities were measured in cell protein extracts using a chemiluminescent assay. c-IAP2, c-IAP1, FLIP-L and β-actin were detected in additional cellular extracts from the same experimental samples by western blotting using specific antibodies applied successively to the same blotted membrane (lower panel). The stars indicate a statistical difference from respective controls (p < 0.05). B) Western blot analysis of caspase-8 cleavage in HeLa cells treated with control compound or RMT 5265 (200 nM for 48 h) with or without poly(I:C) (1 μg/ml; last 16 h). In order to provide a positive control for caspase 8 cleavage, HeLa cells were treated with TNF-α (50 ng/ml) plus cycloheximide (2.5 μg/ml) for 6 h. The same blotted membrane was stained with anti-tubulin-α for protein loading control.