Figure 5
Effect of RVL on B16M cell response to IL-18. B16M cells, either untreated or pre-treated for 30 minutes with 2.5 μM RVL, were incubated with 1 ng/ml IL-18 for different time periods. A) Effect of RVL on B16M-Luc cell retention in hepatic microvasculature. Mice were intrasplenically-injected with either treated or untreated B16M-Luc cells and livers were analyzed after 18 hours. Light emission values were determined as described in Methods. The number of arrested B16M-Luc cells was calculated on the basis of a standard curve relating specific relative light units to B16M-Luc cell number. B) Effect of RVL on B16M cell adhesion to HSE. The percentage of adherent B16M cells was determined as described in Material and Methods. C) Effect of RVL on B16M cell proliferation. B16M cell proliferation was analyzed by sulforhodamine-101-based fluorometric assay. D) Effect of RVL on H2O2 production by B16M-Cells. H2O2 production was expressed as DCF fluorescence values (in fluorescence arbitrary units). E and F) Inhibitory effect of Resveratrol on IL-18-induced NF-κB activation. B16M cells were serum-starved for 24 h and treated with IL-18 (100 ng/ml) from 30- to-120 min. In some experiments, cells received 2.5 μM RVL 30 min before assays. Results shown are representative of the experiment after 60 min of treatment. NF-κB nuclear translocation was detected by immunofluorescent staining with anti p-65 antibody. (E). Western analysis was also performed with anti-p65 antibody and Lamin B as a loading control for the nuclear fraction (F). Every assay was done in triplicate and repeated three times. Data represent average values ± SD. *Differences were statistically significant with respect to cells in basal medium (P < 0.01) according ANOVA test.