FIG. 2.

pRb-mediated senescence is linked to p27^KIP1 accumulation. SAOS-2 cells were transfected with empty vector (pSVE), RB, HA-p107, or HA-p130 expression plasmids and pBabe-puro to select transfected cells by puromycin drug resistance. (A) Forty-eight hours after transfection cells were placed under puromycin selection. Five days posttransfection cells were labeled with BrdU and immunostained with an anti-BrdU antibody (Roche), and BrdU-positive cells were counted (at least 250 cells). The number of BrdU-positive cells is represented as a decrease in BrdU-positive cells compared to vector-transfected cells and represents the average of at least three experiments. (B) Forty-eight hours posttransfection cells were selected with puromycin and then 10 days after transfection were stained for SA–β-gal activity and flat and SA–β-gal-positive cells were counted. Percent flat and SA–β-gal-positive cells indicates the number of flat cells or SA–β-gal-positive cells divided by the total number of cells counted (at least 100 cells) and represents at least five independent experiments. (C) Immunoblot of cells 2 and 10 days posttransfection. (D) p27^KIP1 expression in senescent cells. Ten days after transfection puromycin-selected cells were immunostained with anti-p27^KIP1.