Figure 4. Rosiglitazone inhibits expression of α4 nAChR through p53.

A, Cellular protein was isolated from H1838 cells treated with increasing concentrations of rosiglitazone as indicated for 24 h followed by Western blot analysis for p53 protein using an anti-p53 antibody. Actin served as internal control for normalization purposes. B, Cellular protein (20 μg) was isolated from H1838 cells were with control or p53 siRNA (100 nM each) for 40 h before exposure of the cells to rosiglitazone (Rosig.) for an additional 24 h. Afterwards, Western blot analysis was performed to detect the p53 and α4 nAChR proteins. Actin was used for loading control for normalization purpose. Con, indicates untreated control cells. C, Cellular protein was isolated from H1838 cells cultured for up to 2 h in the presence or absence of SB239023 (10 μM), PD98059 (25 μM) before exposure of cells to rosiglitazone (Rosig., 10 μM) for an additional 24 h, then subjected to Western Blot analysis for p53 protein. Actin served as internal control for normalization purposes. Con, indicates untreated control cells. D, H1838 cells were transfected with a mixture of inducible p53-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1, 0.1μg/μl) for 24 h, then treated with SB239023 (10 μM), PD98059 (25 μM) for 1 h before exposure of the cells to rosiglitazone (Rosig., 10 μM) for an additional 24 h. The ratio of firefly luciferase to renilla luciferase activity was quantified as described in Material and Methods. The bars represent the mean ± SD of at least four independent experiments for each condition. * indicates significant increase of activity as compared to controls. ** indicates significance of combination treatment as compared with rosiglitazone (Rosig.) alone (P < 0.05). Con, indicates untreated control cells.