MAL knockdown alters actin polymerization, terminal maturation, and proplatelet formation of MKs. CD34⁺ cells isolated from cord blood or cytapheresis were transduced with the control lentivirus or the lentivirus encoding shRNA of MAL (shMAL). (A) GFP⁺ cells (day 8) were allowed to adhere on collagen I or convulxin for 2 hours. Stress fibers, filopodia, and lamellipodia were stained with TRITC-conjugated phalloidin (red) and nucleus with 4,6-diamidino-2-phenylindole (blue). The percentage of MK-forming stress fibers, filopodia, or lamellipodia was evaluated on a total of 500 cells using fluorescent light microscopy (original magnification ×40). (B) Ultrastructural aspect of control (i) and shMAL (ii-iii) transduced MKs. MKs were sorted at day 10 of culture on the expression of GFP and fixed. N indicates nucleus; DM, demarcation membranes. Arrowhead represents α granules. (i) Morphology of a typical normal MK. Bar represents 5 μm. (ii-iii) Morphology of shMAL-transduced MK. (ii) Bar represents 2 μm. (iii) Bar represents 5 μm. (C) GFP⁺ cells were sorted at day 9 on the coexpression of CD41 and CD42. Cells were seeded at 2 × 10³ cells/well in a 96-well plate. The percentage of proplatelet-forming MKs was estimated by counting MKs exhibiting one or more cytoplasmic processes with areas of constriction. A total of 500 cells per well were counted at day 13 for cytapheresis-derived MKs and at day 15 for cord blood–derived MKs. Error bars in histograms represent the SD of one representative experiment performed in triplicate wells. Similar results were obtained in 3 repeated experiments with cytapheresis and 2 experiments with cord blood. (D) MKs derived from cytapheresis were plated as in panel C, and the area of proplatelet network estimated in microns squared was measured using Axio Vision 4.6 software. One representative control- and shMAL-transduced proplatelet-forming MKs is shown on left, and the area mean of 10 cells from each group is shown in the histogram on right. Error bars in histograms represent the SD obtained for 10 cells in one representative experiment (n = 2).