siRNA-mediated silencing of TEL/AML1 induces apoptosis in REH cells. (A) Schematic representation of the siRNA sequences (C1, functional; S, control) and their localization within the TEL/AML1 translocation breakpoint mRNA. (C1, position at bp 15-33, and S, at bp 6-24, accession no. S78496). (B) Time course of siRNA transfection, TEL/AML1 depletion, and induction of apoptosis. (C) Depletion of TEL/AML1 after 2 transfections with siRNA C1. Western blot analysis was performed from REH cell lysates treated with the control siRNA S (lane 2) or functional siRNA C1 (lane 3) or untreated (lane 1) at day 6 of the experiment. TEL/AML1 protein was detected using an anti-TEL antibody (kindly provided by P. Marynen, University of Leuven, Leuven, Belgium) and an antitubulin antibody (Oncogene/EMD Biosciences, San Diego, CA) for loading control. (D) Apoptosis was assessed by FACS analysis of annexin V–FITC/PI staining of untreated, siRNA S–treated (control), and siRNA C1–treated (functional) cells at day 8 of the experiment. Data are shown from 1 of 4 representative experiments and indicate early (annexin V single-positive cells) and late (annexin V and PI–positive cells) apoptosis. The difference in annexin V positivity of REH cells treated with the siRNA C1 compared with either control was significant (P < .03 and P < .01), while the 2 controls did not differ significantly between each other (P = .07).