KLF4 inhibits growth of BL cell lines. (A) Expression of KLF4-ER protein in stable clones of BL cell lines Ramos and Namalwa was measured by immunobloting using anti-KLF4 antibody. Anti-ACTB antibody was used as a loading control. (B) Cell lines were plated in 6-well plates at a density of 0.5 × 105 cells per well in 3 mL of complete culture medium (day 0). 4-OHT was added at concentration 200nM at the days 0, 2, and 4. Cells were counted with a hemocytometer. Cell viability was controlled by trypan blue staining. All experiments were repeated at least 3 times. The data represent mean ± SD of the most representative experiment. (C-D) Cell-cycle analysis. Cells were seeded at the density of 1 × 106/10 mL of the complete medium and were treated with 200nM 4-OHT at the same day; in 48 hours, cells were harvested and cell-cycle distribution was measured by PI staining as described in “Cell proliferation assay, apoptosis, and cell-cycle analysis.” Data are mean ± SD obtained in 3 independent experiments. (E) Cells were seeded at concentration of 1 × 106 cells per 10 mL of complete medium. The 4-OHT was added at the same concentration and schedule as for cell proliferation experiment and cell-cycle experiments. Cell death was measured by annexin V/PI staining. The results are represented as specific apoptosis (SA): SA (%) = 100(AE − AC)/(1 − AC), where AE equals percentage of apoptotic cells in the experimental (+4-OHT) group and AC equals percentage of apoptotic cells in the control (not treated) group. Data are mean ± SD of 1 most representative of at least 3 independent experiments.
