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.