Effect of vorinostat inhibits STAT6 in HL cell lines. (A) L-428 and KM-H2 cell lines were incubated with 5 μM of vorinostat for 6 to 48 hours. Whole cell lysates were examined by Western blotting. As shown, vorinostat induced down-regulation of pAkt (Ser473) without inducing changes in total Akt or pAkt (Thr308), and decreased STAT6 and pSTAT6 levels without a significant effect on STAT3. (B) Vorinostat down-regulated STAT6 mRNA expression in a dose-dependent manner. Cells were cultured with medium and vorinostat (1-5 μM) for 24 hours, and the effects on STAT6 mRNA expression was determined by the TaqMan PCR assay. Results were normalized to GAPDH and represent the mean of 3 independent experiments (± SEM). (C) L428 cell line was transfected with STAT6 siRNA and the efficacy of this method was confirmed by measuring STAT6 protein levels by Western blot. Cells that were incubated with STAT6 siRNA demonstrated a 50% reduction in STAT6 protein after 48 hours compared with those incubated with control siRNA. This is a representative experiment of 3 independent experiments demonstrating similar results. (D) Effect of STAT6 knock down by siRNA on cell proliferation. L-428 cells that were transfected with STAT6 siRNA demonstrated lower proliferative rate and thymidine incorporation compared with those transfected with control siRNA.