FOXO1 induces growth arrest and apoptosis in cHL cell lines. (A) Expression of FOXO1(A3)ER in clones of cHL cell lines. Western immunoblot using antibodies specific for FOXO1 and ACTB as loading control are shown. (B) Growth-inhibitory effect of FOXO1. cHL cell lines stably expressing the empty vector or the vector containing FOXO1(A3)ER were seeded in a 6-well plate in 2 mL of complete culture medium at a density of 1 × 105 cells/well and treated with 4-OHT at a concentration of 200nM or with a vehicle. Numbers of live cells were calculated by hemacytometer using trypan blue staining (C). Apoptosis was measured by annexin V–PE/7-amino-actinomycin D staining at the same time points as cell counting. Data represent the mean ± SD of 3 independent experiments. (D) FOXO1 activation inhibits cell-cycle transition. A total of 1 × 106 cells were seeded in 10 mL of complete medium. The experimental group was treated with 200nM 4-OHT. After 24 hours of incubation with 4-OHT, cells were harvested and cell-cycle distribution was analyzed by propidium iodide. Bars represent the mean of 3 measurements ± SD. The data are representative of at least 3 independent experiments that gave similar results.
