p21 silencing in CLL cells interferes with the antiproliferative activity of lenalidomide. (A-B) siRNAs specific for p21 or nonspecific siRNA control (CTRL) were transfected into CLL cells using HiPerFect reagent as described in “Methods,” and cocultured on HeLa_CD154 and IL-4/IL-10. After 48 hours, the cells were collected and lysed for detection of p21 and GAPDH protein by immunoblot. In (A), data from 2 representative patients are presented, and in (B) densitometry analysis quantifying the levels of p21 protein in 5 different CLL samples is shown. The expression of p21 has been normalized to GAPDH. **P < .01 (Student t test, mean ± SEM; n = 5). (C-D) CLL cells from 4 patient samples were transfected as in (A) with either CTRL siRNA or p21 siRNA, and cocultured on HeLa_CD154 and IL-4/IL-10 in the presence of 3 µM lenalidomide or control media. After 48 hours, the cells were collected and lysed for detection of p21 and GAPDH protein by immunoblot. In (C), data from 2 representative patients are presented, and in (D) densitometry analysis quantifying the levels of p21 protein in 4 different CLL samples is shown. The expression of p21 has been normalized to GAPDH. *P < .05 (Student t test, mean ± SEM; n = 4). (E) CLL cells from 5 patient samples were transfected as in (A) with either CTRL siRNA or p21 siRNA, and exposed to 3 µM lenalidomide or DMSO. After 72 hours, CLL-cell proliferation was measured using 5-bromo-2′-deoxyuridine (BrdU) incorporation, which provides absorbances at 450 to 690 nm. *P < .05; **P < .01 (Student t test, mean ± SEM; n = 5). (F) Proliferation data from (E) were used to calculate the percent inhibition of proliferation induced by lenalidomide in p21-silenced cells and in CTRL cells ([Absorbance_{CTRL-treated samples} – Absorbance_{lenalidomide-treated samples}] / Absorbance_{CTRL-treated samples} × 100). **P < .01 (Student t test).