Clustering and characterization of small-molecule inhibitors of the cyclin D2 promoter. (A) The numeric value of the inhibition of cyclin D2 transactivation was represented colorimetrically. Drugs were assigned into families based on the annotation from the LOPAC and Prestwick libraries and clustered using the Cluster and Treeview algorithms.18 The results of the representative reproducible hits are shown. To determine whether molecules identified in the screen were nonspecific inhibitors of luciferase, preferential inhibitors of c-maf–dependent cyclin D2 transactivation, or c-maf–independent inhibitors of cyclin D2 transactivation, hits were tested in NIH3T3 cells overexpressing the RSV promoter driving luciferase, NIH3T3 cells overexpressing c-maf and the cyclin D2 promoter driving luciferase, and NIH3T3 cells without c-maf but overexpressing the cyclin D2 promoter driving luciferase. Molecules were tested at a final concentration of 5 μmol/L. Selected glucocorticoids were tested in dose-response studies in NIH3T3 cells overexpressing c-maf and the cyclin D2 promoter driving luciferase. The mean IC50(± SD) is shown, where the IC50 represents the concentration of the compound required to reduce luciferase activity by 50% from untreated control cells. (B) NIH3T3 cells overexpressing c-maf were treated with selected glucocorticoids (final concentration 5 μmol/L). Twenty-four hours after treatment, cell lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted using antibodies specific for c-maf and β-actin.
