BAK1 activation is responsible for KLF4-induced apoptosis in cHL cell lines. (A) KLF4 induces BAK1 mRNA expression. A total of 2 × 10⁶ KMH2, L428, Namalwa, and Ramos cells stably expressing KLF4-ER fusion protein, or empty vector were seeded in 10 mL of complete culture medium and treated with 200nM of 4-OHT at the same day. Twenty-four hours later, cells were harvested and BAK1 expression was assessed by quantitative PCR. Data are relative mRNA expression (mean ± SD) calculated by the comparative C_t method. The relevant nontreated controls were used as comparators. RPL13A was used as reference gene. All experiments were performed in triplicate. (B) KLF4 activation induces BAK1 protein expression in cHL cell lines. Western immunoblots of transfected cells with or without 4-OHT are shown. ACTB was used as loading control. (C-D) Silencing of BAK1 expression by shRNA prevents KLF4 induced cell death. (C) KM-H2-KLF4-ER or L428-KLF4-ER cells were transiently transfected with pRS-luci and pRS-shBAK1 vectors followed by 5-day selection with 0.5 μg/mL (KM-H2) and 1 μg/mL (L428) of puromycin. BAK1 expression was determined by immunoblot. (D) KM-H2-KLF4-ER and L428-KLF4-ER were transfected with pRS-BAK1 or pRS-luci and were selected for 5 days with puromycin. Then cells were recovered for 1 day in the complete culture medium without puromycin followed by treatment with 200nM of 4-OHT (day 0). Apoptosis was measured on the indicated time points. Data are mean ± SD obtained in 2 independent experiments.