Figure 5

The effect of cafestol and kahweol on downstream target proteins by the specificity protein 1 (Sp1). The firefly luciferase Cyclin D1(A), Mcl-1 (B), or Survivin (C) reporter gene activity was assessed. For the reporter gene assay, MSTO-211H was transfected with a plasmid mixture containing the Cyclin D1, mcl-1, or survivin luciferase reporter gene (250 ng) and the β-gal gene (20 ng) for normalization. At 24 hours after transfection, cells were treated with various concentrations of cafestol or kaweol for 48 hours. Results are shown as the mean ± SD of triplicate samples from three independent experiments. The asterisks (*, P < 0.05) indicated a significant change relative to cafestol or kahweol-treated cells compared with untreated cells. The effect of cafestol (D) and kahweol (E) for 48 hours on Cyclin D1, Mcl-1, and Survivin was determined by Western blots. Fifty micrograms of cellular extract per lane were separated on the SDS-PAGE gel as described in materials and methods. Equal loading of protein was confirmed by incubating the same membrane with an anti-β-Actin antibody.