Inhibition of TWIST in KG1a cells and its effect on apoptosis. Early stage apoptosis (annexin V⁺, propidium iodide⁻) in KG1a and PL-21 cell lines transfected with either a scrambled siRNA sequence (SCR) or siRNA specific for TWIST (siTWIST), and exposed overnight to normal medium (veh) versus exogenous TNFα (25 ng/mL, A) or TRAIL (100 ng/mL, B), respectively. Apoptosis was determined by flow cytometry. (C) Cell preparation as in panels A and B, but cultured in normal medium (veh) or in the presence of the soluble TNF receptor etanercept (10 μg/mL for 24 hours [concentration based on ancillary studies]). Error bars indicate SEM. (D) Protein lysates from the same cell preparations were separated on 4% to 12% Bis-Tris gels and immunoblotted with antibodies against TWIST, p53-ser46, Bax, and caspase 9, respectively. β-Actin served as loading control. (E) TWIST and p53 levels in primary CD34⁺ MDS cells in which TWIST was silenced by specific siRNA (KD TWIST) compared with levels in unmodified cells from the same primary CD34⁺ MDS sample.