Figure 3

ETS1 binding and regulation of MMP9 promoter transcription in HMC-3A, A253, and iSGECs. (A) HMC-3A (E6) and control HMC-3A (EM) were transiently transfected with either 250 ng of pGL3-basic MMP9 promoter full length (−923 bp to + 18 bp) or 5′-deletion MMP9 promoter constructs and 30 ng of control plasmid expressing renilla luciferase under the control of thymidine kinase promoter (pRL-TK). (B) Results were expressed as fold increase in RLU over the control normalized to pGL3-basic MMP9 expression. 5′-deletion constructs demonstrated significant increases in luciferase activity within the −366 to −216 promoter region of the MMP9 promoter. (C,D) EBS-MUT1/2/5 sites displayed significant reductions in luciferase activity compared to the full length −366 bp-MMP9-pGL3 promoter plasmid. (E) Activity of the—366 bp-MMP9-pGL3 ETS1 binding site mutants (EBS-MUT 1, 2, 1 + 2, and 5) were further investigated by transient transfection into A253 (n = 6), iSGEC-nSS2 (n = 6), and iSGEC-pSS1 (n = 4). (F) ChIP-qPCR assay performed with SGCLs and iSGECs was utilized to assess the functional relevance of ETS1 interaction with the MMP9 promoter spanning across EBSMUT1,2, and 5. Cell lysates immunoprecipitated with normal mouse IgG (negative control) (blue) or ETS1 (red) antibodies. Samples were normalized to 5% of sample input. Significant comparisons among control and ETS1 antibody were made by Mann–Whitney U-test with p values indicated over their respective bars (***p < 0.001), (**p < 0.01), (*p < 0.05). Error bars represent mean +/− standard deviation (SD).