The effect of miR-221 on p27^Kip1 expression and cell apoptosis. (A) p27^kip1 expression during monocyte differentiation into imDCs and mDCs. p27^kip1 expression in human monocytes (Mo) at day 0 (D0), imDCs at days 2, 4, and 6, and mDCs at day 8 was analyzed by Western blot. (B) miR-221 expression during monocyte differentiation into imDCs and mDCs by real-time PCR analysis. (C) Increased expression of p27^kip1 after silencing of miR-221. imDCs at day 4 were transfected with miR-221 knockdown probe (Anti-miR-221) or scramble control (Scramble) at the final concentration of 40nM for the indicated time. Cells were then analyzed for p27^kip1 expression by Western blot. (D) Increased apoptosis after silencing of miR-221. imDCs at day 2 or day 4 (data not shown) were transfected with miR-221 knockdown probes or scramble controls (40nM) for 40 hours. Cells were then stained with APC-conjugated annexin V and analyzed on a FACSCalibur. The data shown in every panel are representative of 2 (B-D) or 3 (A) independent experiments.