Figure 3.

Overexpression of cyclin D1 activates both p14^ARF and p16^INK4a in human mammary epithelial cells (HMECs). A: HMECs were infected with lentivirus expressing doxycycline (Dox)-induced cyclin D1. After puromycin selection, 0.5 μg/mL Dox was added, and cells were harvested after 0, 24, 48, and 96 hours, respectively. p14^ARF (light gray bars) and p16^INK4a (dark gray bars) mRNA levels measured by real-time PCR using β-actin as a control. Cyclin D1 and p14^ARFexpression were analyzed by immunoblots with β-actin as a loading control. B: HMECs were manipulated to coexpress control or DMP1 shRNA (Ctrl) and cyclin D1 (D1) or D1T286A (D1TA) as indicated. Real-time PCR analysis of p14^ARF (light gray bars) and p16^INK4a (dark gray bars) mRNA levels after normalized against β-actin. Immunoblot analysis of Dmp1 and HA-tagged cyclin D1 expression. β-Actin was used as loading control. The asterisk indicates nonspecific bands. C: HMEC cells were treated as in B and stained with propidium iodide followed by flow cytometric analysis (top panel). Quantification of cell-cycle distribution is shown in the bottom panel.