MAL/SRF complex is involved in platelet formation and megakaryocyte migration by regulating MYL9 (MLC2) and MMP9 by Laure Gilles, Dominique Bluteau, Siham Boukour, Yunhua Chang, Yanyan Zhang, Thomas Robert, Philippe Dessen, Najet Debili, Olivier A. Bernard, William Vainchenker, and Hana Raslova Blood Volume 114(19):4221-4232 November 5, 2009 ©2009 by American Society of Hematology

MAL expression in MKs. CD34+ cells from cord blood or cytapheresis were cultured with TPO. (A) At day 9 of culture, mRNA levels of MAL and MAL16 were evaluated by quantitative RT-PCR. MAL expression in MKs. CD34+ cells from cord blood or cytapheresis were cultured with TPO. (A) At day 9 of culture, mRNA levels of MAL and MAL16 were evaluated by quantitative RT-PCR. Their expression was normalized with respect to HPRT mRNA. (B) To investigate MAL transcription level during MK differentiation, cells were sorted at day 6 of culture in 4 populations: for cord blood, CD34+CD41−, CD34−CD41+CD42−, CD34−CD41+CD42low, and CD34−CD41+CD42high; and for cytapheresis, CD34+CD41−, CD34+CD41+CD42−, CD34+CD41+CD42+, and CD34−CD41+CD42+. MAL mRNA level was evaluated as in panel A. Error bars in panels A and B represent the SD of the mean of 3 repeated experiments each performed in triplicate wells. (C) To investigate protein levels by Western blot analysis during MK differentiation, total cell populations were harvested on day 3 (D3), day 6 (D6), day 9 (D9), and day 12 (D12) for cord blood-derived MK and at day 0 (D0), day 3 (D3), day 6 (D6), and day 9 (D9) for cytapheresis-derived MKs. HDAC-1 and HSC70 were used as internal control. The figure illustrates representative data of 2 experiments with similar results. Laure Gilles et al. Blood 2009;114:4221-4232 ©2009 by American Society of Hematology

MAL nuclear localization after Rho GTPase activation in MKs MAL nuclear localization after Rho GTPase activation in MKs. (A) MAL cytoplasmic localization in serum-starved NIH3T3 cells and its nuclear localization after serum stimulation. MAL nuclear localization after Rho GTPase activation in MKs. (A) MAL cytoplasmic localization in serum-starved NIH3T3 cells and its nuclear localization after serum stimulation. (B) Cord blood-derived MKs (upper panels) and cytapheresis-derived MKs (lower panels). After adhesion to polylysine for 10 minutes (PLL, 10′), MAL is localized in the cytoplasm. After adhesion to collagen I or convulxin for 2 hours, MAL is localized in the nucleus. Both substrates induce Rho GTPase activation. MAL (red) and 4,6-diamidino-2-phenylindole (blue) staining was detected by immunofluorescence and visualized under a fluorescent light microscope at an original magnification ×60. Laure Gilles et al. Blood 2009;114:4221-4232 ©2009 by American Society of Hematology

Effect of MAL knockdown on ploidy level and MK differentiation. 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. Laure Gilles et al. Blood 2009;114:4221-4232 ©2009 by American Society of Hematology

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). 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). Laure Gilles et al. Blood 2009;114:4221-4232 ©2009 by American Society of Hematology

Gene profiling of MKs after MAL knockdown. Gene profiling of MKs after MAL knockdown. CD34+ cells isolated from cord blood and cytapheresis were transduced with the control lentivirus or the lentivirus-expressing shMAL. At day 9 of culture, cells expressing GFP were sorted and mRNA was subjected to microarray analyses using Agilent Human Whole Genome 44K oligonucleotide arrays (A) and to quantitative RT-PCR (B). (A) A total of 1929 and 1436 probe sets were found significantly deregulated in MKs derived from cytapheresis and cord blood, respectively, with a 613 probe set common to both. The table shows primary sequence name, accession number, and fold change of the 20 most down-regulated genes after MAL knockdown in cytapheresis- and cord blood-derived MKs. (B) Validation of microarray data (i) by quantitative RT-PCR (ii) for 12 selected genes. The error bars represent the SD of the mean of 3 repeated experiments each performed in triplicate wells. Laure Gilles et al. Blood 2009;114:4221-4232 ©2009 by American Society of Hematology

MMP9 and MYL9 are directly regulated by MAL/SRF complex. MMP9 and MYL9 are directly regulated by MAL/SRF complex. (A) Schematic representation of MMP9 and MYL9 human promoter regions cloned into the pLuc-MCS reporter. Arrows represent the translation start site. (B) Luciferase assay performed by transient transfection of HEK293 cells with the 50 ng of Megix vector containing MAL. Luciferase levels are shown as fold change relative to cells transfected with the reporter construct alone. The total amount of transfected DNA was kept constant by addition of empty Megix vector. The histogram shows one representative experiment of 3, each in triplicate. Error bars represent the SD of triplicate. (C) ChIP assay performed in cytapheresis-derived MKs (day 10 in culture) with primer sets directed toward in silico predicted SRF-binding sites: MMP9_C_F and R, and MMP9_D_F and R for MMP9, and primer sets MYL9_A_F and MYL9_E_R for MYL9. Localization of primers MMP9_C_F and R for MMP9 and MYL9_A_F and MYL9_E_R for MYL9 are depicted in panel A. Primers MMP9_D_F and R are not localized in the cloned promoter region designed in panel A. Control primer sets allowing amplification of known SRF-binding sites (THSB1) or DNA region devoid of SRF sites were also used. Immunoprecipitation was performed with control rabbit IgG and anti-SRF antibodies. Histograms indicate relative occupancy of SRF-binding sites by SRF in the MMP9, MYL9, and THSB1 promoters. Error bars represent the SD of experiments performed in duplicate. The figure illustrates representative data of 2 independent experiments with similar results. Laure Gilles et al. Blood 2009;114:4221-4232 ©2009 by American Society of Hematology

MAL contribution to proplatelet formation and migration of MKs by targeting MYL9 and MMP9, respectively. 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. Laure Gilles et al. Blood 2009;114:4221-4232 ©2009 by American Society of Hematology