Figure 2. Downregulation of cyclin D1 expression mediated the inhibition of colony formation and induction of G0/G1 phase arrest following ISO treatment.

(A-C) Cl41 cells were pretreated with indicated doses of ISO for 30 min and then co-treated with ISO and TPA/EGF (40 ng /ml) for 24 hours (A) or pretreated with 50 μM ISO for 30 min and then co-incubated with ISO and TPA/EGF for the indicated time periods (B and C). The protein expression levels were determined by Western blot, and β-actin levels were used to as protein loading control. Data was representative one of three independent experiments. (D and E) The FLAG-cyclin D1 expression levels were determined by Western blot in Cl41 cells transfected with plasmid encoding FLAG-tagged cyclin D1 T286A mutant and its vector control. Antibodies specifically against cyclin D1 and FLAG were used, respectively, to detect the protein as indicated in the figure (D). Transfectants were treated with TPA/EGF (40 ng /ml) alone, or pretreated with 50 μM ISO for 30 min and then co-incubated with ISO and TPA/EGF for 24 hours. Antibody specifically against cyclin D1 was used to determine cyclin D1 expression (E). (F and G) Cl41 cells were treated with TPA/EGF (40 ng /ml) alone, or pretreated with 50 μM ISO for 30 min and then co-incubated with ISO and TPA/EGF for 24 hours. Cell cycle distribution was determined by flow cytometric analysis. Data represent one of three different experiments. (H and I) Cells stably transfected with vector control or FLAG-cyclinD1 T286A were treated with medium containing either DMSO or TPA/EGF (40 ng /ml) with or without exposure to ISO (50 μM) for 3 weeks. Representative images of colonies of transfectants in soft agar assay were presented (I). Colonies were counted after 3 weeks and the results were presented as colonies per 10,000 cells from three independent experiments (H). The asterisk (*) indicates a significant difference of colony number between Cl41 FLAG-cyclinD1 T286A cells and Cl41 vector cells (P<0.05).