MAL contribution to proplatelet formation and migration of MKs by targeting MYL9 and MMP9, respectively. (A-B) CD34+ cells isolated from cytapheresis were transduced at days 1 and 2 of culture with the lentivirus-encoding control shRNA (control) or MYL9 shRNA (shMYL9). (A) GFP+ cells were analyzed at day 9 of culture. MYL9 mRNAs were measured by quantitative RT-PCR (left panel). The histogram shows one of 2 representative experiments, each in triplicate. Error bars represent the SD of triplicate wells. (B) The percentage of proplatelet-forming MKs derived from cytapheresis samples was evaluated as described in Figure 4C at day 13 of culture. Error bars represent the SD of 1 representative experiment performed in triplicate wells. Similar results were obtained in 4 independent experiments. (C) MKs derived from cytapheresis CD34+ cells 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 shMYL9-transduced proplatelet-forming MKs is shown on the left, and the mean area of 10 cells from each group is shown in the histogram on the right. Error bars in histograms represent the SD obtained for 10 cells in 1 representative experiment (n = 2). (D) MK migration through Matrigel-coated transwells in response to SDF-1. Cytapheresis isolated CD34+ cells were transduced at days 1 and 2 of culture with a control or shMAL-encoding lentivirus. The experiment was done on day 8 of culture. Data represent the percentage of migrated CD41+CD42+GFP+ cells compared with total CD41+CD42+GFP+ input. Error bars represent the SD of 1 representative experiment performed in triplicate. The figure illustrates representative data of 2 independent experiments with similar results.