Figure 13

AAV2 infection of different breast cancer cell lines and induction of apoptosis/cell death results in altered expression of proliferation markers PCNA, Ki67 and c-Myc. (A) MCF-7 (B) MDA-MB-468 (C) MDA-MB-231 and (D) CIN-612 9E (HPV31b positive cervical cancer cells) monolayer cell cultures were synchronized by trypsinization of 80% confluent cultures and plating at a density of 1 × 10⁶ cells in E medium. The cells were incubated for 10 to 12 h at which point at least two-thirds of the cells are maximally synchronized in the G1 phase followed by infection with AAV2. This time point was designated time zero (t = 0). Cell pellets were collected each day over a 7 day period. Cells were passaged 1:2 on day 2 and day 5. Detection of cell cycle proliferation proteins was performed by Western blotting. Total protein extracts were prepared as described previously [48]. Sixty micrograms of total protein extracts from AAV2 infected and control cells were resolved in SDS-polyacrylamide gel electrophoresis (PAGE) gels. The PCNA protein was detected using a rabbit polyclonal antibody (Santa Cruz). The Ki67 protein was detected using a rabbit polyclonal antibody (Santa Cruz). The c-Myc protein was detected using a rabbit polyclonal antibody (Santa Cruz).