Figure 1

TSA suppresses PMA-induced OPN transcription in HeLa cells. A. HeLa cells were preincubated with 0-1 μM TSA for 1 h followed by treatment with PMA (50 ng/ml) for 6 h. Whole cell lysates were analyzed by western blot using anti-OPN antibody. B. HeLa cells were pretreated with TSA followed by PMA under similar conditions as described above. Total RNA was isolated and OPN mRNA levels were detected by semiquantitative RT-PCR and analyzed by agarose gel electrophoresis. Actin was used as control. The data represents three experiments exhibiting similar results. C. HeLa cells were cotransfected with hOPN promoter deletion constructs (-500/+20, -267/+20, -127/+20, -70/+20 and -20/+20) containing luciferase reporter gene along with renilla luciferase vector, pRL followed by stimulation with PMA. The luciferase activity was measured in cell lysates and normalized to Renilla luciferase activity. Fold-changes in luciferase activity with respect to control were calculated. Columns, mean of triplicate determinations; bar, S.D. *, p < 0.05. D. HeLa cells were cotransfected with various hOPN promoter mutation constructs (-500/+20, CM, AM and OM) containing luciferase gene along with pRL vector and treated with PMA. The luciferase activity was measured and normalized. Fold-changes in luciferase activity with respect to control were calculated. Columns, mean of triplicate determinations; bar, S.D. *, p < 0.05.