Fig. 3

Inhibition of cyclin D1 transcription by TC-HW through the inhibition of Wnt activation. a and b HCT116 and SW480 cells were treated with TC-HW at the indicated concentrations for 24 h. For Western blot analysis (a), cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against β-catenin or TCF4. Actin was used as internal control for Western blot analysis. For RT-PCR analysis of the gene expression of β-catenin and TCF4 (b), total RNA was prepared. GAPDH was used as internal control for RP-PCR. c HCT116 and SW480 cells were co-transfected with TOP-FLASH or FOP-FLASH constructs containing wild-type or mutated TCF binding sites and pRL-null. The cells were treated with TC-HW for 24 h. Luciferase activity for TOP-FLASH and FOP-FLASH was measured as a ratio of firefly luciferase signal/renilla luciferase signal using a dual luciferase assay kit. *P < 0.05 compared to cell without TC-HW