Figure 2.
SiRNA-mediated lef-1 knockdown leads to apoptosis in primary CLL cells and growth inhibition in JVM-3 cell line. Freshly isolated CLL cells were nontransfected (NT), mock-transfected (no siRNA), transfected with 0.5 µM control nontargeting or lef-1 siRNA (n = 6), and cultured for 16 hours in complete medium. (A) Cell viability was evaluated by Annexin V/PI staining, and the results from each patient are presented as the percentage of Annexin V-/PI- cells (*P < .05). (B) SiRNA-mediated lef-1 knockdown was carried out in JVM-3 cell lines. Forty-eight hours after nucleofection, protein extracts were analyzed for lef-1, c-myc, cyclin D1, β-catenin, PCNA, and cleaved PARP by immunoblot analysis, with β-actin as the loading control. There was down-regulation of lef-1 target genes along with PCNA and PARP, indicating growth inhibition of the cell line. These observations were confirmed in two independent experiments.