AAV2 induced DNA laddering/degradation in multiple breast cancer cell lines. (A) MCF-7, (B) MDA-MB-468, (C) MDA-MB-231 and (D) nHMECs monolayer cultures were synchronized in G1, followed by infection with AAV2 at an MOI of 0.02. Cell pellets were collected each day over a seven day period for the breast cancer cell lines, and over a five day period for the nHMECs. The breast cancer cell lines were passaged 1:2 on day 2 and day 5, and on day 2 for nHMECs. DNA laddering assays were performed by isolating low-molecular weight DNA using protocols described herein. Twenty micrograms of DNA were resolved in a 1% agarose Tris-Borate-EDTA gel and stained with ethidium bromide. Results shown are representative of three individual experiments. t, time; +, AAV2 infected; -, control. DNA ladders are indicate the following base-pair fragments starting at the top: 23941, 9416, 6557,4361, 2322, 2027, 1353, 1073, 872, 603, 310. (E) nHMECs cultured from multiple patient breast tissue biopsies were used to prepare whole cell extracts. Five micrograms of the extract was used for western blot analysis. Cytokeratin markers Involucrin, K10 and K14 were detected using antibodies as described in Materials and Methods.