FLLL32 reduced STAT3 DNA binding and gene expression. (A) STAT3 DNA binding was measured in A375 cells following a 16 hour treatment with FLLL32 (2 μM or 4 μM) as described in the Methods section. Unlabeled target DNA were included to compete for binding as indicated, and a STAT3 specific Ab was used to were included confirm specificity (last lane). Cell lysates were evaluated concurrently by immunoblot to control for total level of STAT3 protein at the 16 hour time point. (B) FLLL32 reduced STAT3-regulated gene expression. Expression of STAT3-regulated genes cyclin D1 and survivin were evaluated following a 24 hour treatment with FLLL32 in melanoma cell lines. Membranes were probed with β actin as a loading control and all blots represent data from at least two independent experiments.