Knockdown of clathrin inhibits enucleation. (A) Human primary erythroid cells differentiated from CD34⁺ cells were transfected on days 10 and 11 with siRNAs against clathrin light chain, or a control siRNA, and the effects on protein expression were determined by Western blot analysis on day 13 (B). On day 14, the cells were collected, washed, cytospun onto poly-L-lysine–coated slides and stained with benzidine and hematoxylin (C), and the percentage of enucleated cells were determined. The percentages of enucleated cells relative to the total number of benzidine-positive cells are shown for 3 independent experiments. Typically, this experimental system yields between 30% and 45% enucleated cells after 14 days. (B) Western blot showing knock-down efficiency of clathrin siRNA. Note that knocking down clathrin light chain decreases the levels of clathrin heavy chain, which is consistent with a previous report.¹⁴  (C) Representative microscopic fields of benzidine and hematoxylin stained cytospins are shown. Orthochromatic erythroblasts and reticulocytes stained positive for benzidine (golden brown). Nuclei stained blue with hematoxylin. Arrows point to reticulocytes. Images were taken with a Leica DM 4000B microscope with 40×/0.75 objectice, visualized with a Leica DFC320 camera and analyzed by Adobe Phototshop 7.0. (D) The total number of live cells in both the siRNA treated conditions were counted by trypan blue exclusion on day 13, and the mean ± standard deviation of 3 independent experiments were plotted.