Figure 5.

Acute loss of Cdh1 expression resulted in the onset of premature senescence in primary human fibroblasts. (A–C) Primary human lung fibroblast cells were infected with the indicated lentiviral shRNA constructs. Twenty-four hours after infection, cells were selected with 1 μg/ml puromycin to eliminate the noninfected cells. Seven days after puromycin selection, cells were fixed and stained for senescence-associated β-Gal activity (A). Six days after puromycin selection, cells were fixed by 70% ethanol and the cell cycle distribution was examined by FACS analysis (B). Six days after puromycin selection, cells were incubated with 1 μg/μl BrdU and 100 μg/μl uridine for 6 h before being fixed with cold methanol and subjected to immunohistochemical analysis using anti-BrdU antibody (C). (D) Quantification of the percentage of BrdU-positive cells in C. (E) Immunoblot analysis of primary human lung fibroblasts (LF1) and SV40 LT-antigen expressing LF1 (LF1/LT) cells infected with the indicated shRNA lentiviral vectors. Twenty-four hours after infection, cells were selected with 1 μg/ml puromycin to eliminate the noninfected cells. Whole-cell lysates were collected at the indicated times after infection.