Fig. 1.

Establishment of ANXA1-reduced cells and assessment of malignant characteristics. (A) Immunoblot of RWPE-1/NC and RWPE-1/rA1 cells. Equal protein loading was verified using antibodies to β-actin (Actin). (B) Proliferation rates were determined by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye conversion after equal numbers of cells were cultured for 72 h in normal growth medium. (C) Anchorage-independent growth was determined by counting colonies >200 μm in diameter that resulted after culture in soft agar for 21 days. Colony-forming efficiency (CFE) of each cell line analyzed is shown. RWPE-2 is a Ras-transformed variant of RWPE-1 (28) and used as a positive control. (D) VP-16 (etoposide)-induced apoptosis was measured using trypan blue exclusion to determine cellular viability after exposure to the indicated concentrations of VP-16 for 48 h. Data are presented as the mean ± SD of three independent experiments.