Exogenous stimuli down-regulate p27 and up-regulate miR221/222 in CLL cells. Protein lysates from purified CLL cells of 1 representative case stimulated with anti-immunoglobulin M (IgM) antibodies, CD40L, CpG-ODN, and the mix of the 3 reagents were subjected to Western blot. Blots were probed with anti-p27 antibody and then stripped and reprobed with β-actin as loading control (A). Mean values of densitometric analysis from 10 different patients are reported (B). Bars represent SE. Asterisks mark significant reduction for CD40L, CpG-ODN, and mix. TaqMan miRNA assay was used to detect miR-221 (C left) and miR-222 (C right) levels in control and stimulated cells. The mean value in 10 patients is reported. The average miR-221 induction was 1.4 ± 0.2 (anti-IgM), 2.5 ± 0.7 (CD40L), 6.1 ± 1.5 (CpG-ODN); and 5.5 ± 1.1 (mix of the 3 stimuli), and the average miR-222 induction was 1.6 ± 0.3 (anti-IgM), 2.3 ± 0.4 (CD40L), 10.6 ± 3.2 (CpG-ODN), and 6.9 ± 1.9 (mix of the 3 stimuli). Bars represent SE. Significance is indicated by asterisks. Statistics were calculated with the Wilcoxon test.