Fig. 5. Effect of Red Wine Extract (RWE) on the HO-1 protein (A) and gene expression (B). (A) Cells were pre-treated with several concentrations of RWE (200, 400 and 600 μg ml^–1) for 30 min and then stimulated with cytokines (20 ng ml^–1 TNF-α; 10 ng ml^–1 IL-1; 50 ng ml^–1 INF-γ). After 24 hours of incubation, total protein extracts were obtained and analysed by western blot using an anti-human HO-1 antibody. In the bar graph, the HO-1 content normalised to the actin level represents the mean ± SEM from at least three independent experiments and is expressed as a percentage of control cells (100%). (B) Cells were pre-treated with RWE (600 μg ml^–1) for 30 min and then stimulated with the mix of cytokines. After 18 hours of incubation, mRNA production was evaluated by qRT-PCR, as described in the “Materials and methods” section, and expressed as fold increase above control cells. Statistical significance: *p < 0.05, **p < 0.005 as compared to control cells; ^&p < 0.05, ^&&p < 0.005 and ^&&&p < 0.001 as compared to cells stimulated with cytokines.