Pharmacologic and siRNA analysis of Sp1/3 activation of <i>Mina</i> promoter activity in EL4 cells.

<p>(A–D) Mapping the key nucleotides required for Sp1/3 binding to each Sp1/3 binding site (Sp1.1–Sp1.4) in the <i>Mina</i> P1 promoter. For each site, gel shift assays were performed with wild type (WT) and mutant (M1–6) probes (comprising an overlapping set of mutations, underlined), as shown to the right of each gel shift assay image. Nucleotides critical for Sp1/3 binding are bolded. Data are representative of 2 independent experiments. (E) All four Sp1/3 binding sites act additively to activate <i>Mina</i> promoter activity. Cloned into a firefly luciferase reporter construct, the four Sp1/3 sites of a WT <i>Mina</i> P1 promoter (spanning position −64 to +151) were mutated individually or in combinations, as indicated (Xs) in the schematic to the left of the bar graph depicting reporter activity. Data are presented as the mean and SEM from 3 independent experiments. Statistical significance was determined by the student’s t-test (ns, not significant; *p<0.05; **p<0.01). <u>Vector</u> is the PGL3 basic vector; <u>SV40 Pro</u> is the PGL3 basic vector containing the SV40 promoter. (F) Effect of Sp1 inhibitor Mithramycin A on <i>Mina</i> transcript level. EL4 cells were treated for 24 hr with 0, 10 nM, 100 nM, 1 uM Mithramycin A or 1 µM DMSO carrier prior to RNA harvest and analysis of <i>Mina</i> mRNA level by quantitative RT-PCR.