Effect of MAL knockdown on ploidy level and MK differentiation. (A) MO7e cells were transduced with a control lentivirus or the lentivirus-encoding shRNA of MAL (shMAL). GFP⁺ cells were sorted and analyzed. MAL mRNA level (left panel) was measured as in Figure 1. MAL protein level (right panel) was analyzed at day 9 of culture by Western blot. HDAC-1 was used as internal control of a quantity. The figure illustrates representative data of 2 experiments with similar results. (B-D) Cord blood and cytapheresis-isolated CD34⁺ cells were transduced with the control lentivirus or the lentivirus-encoding shRNA of MAL (shMAL). (B) MAL protein level was analyzed at day 9 of culture by Western blot in GFP⁺ cells. HDAC-1 and HSC70 were used as internal controls. The figure illustrates representative data of 2 experiments with similar results. (C) The CD41⁺CD42⁺ cell population (left panel), corresponding to mature cytapheresis-derived MKs, was analyzed for ploidy level by Hoechst staining (right panel). The mean ploidy (N) was calculated from the number of cells at each ploidy level. The figure illustrates representative data of 3 experiments with similar results. (D) The percentage of mature MKs was evaluated as the percentage of cells coexpressing both CD41 and CD42 markers. Data illustrate analysis of 4 repeated experiments for cytapheresis and 3 for cord blood. Similar results were obtained in all experiments.