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. 2014 Jul 1;9(7):e101151. doi: 10.1371/journal.pone.0101151

Figure 5. Luciferase reporter assays in HeLa cells demonstrate that Miz-1 activates gene expression via Mizm1.

Figure 5

(A) Four luciferase reporter vectors were constructed: pGL3ec containing no putative Miz-1 binding motifs, pGL3e-MizM containing four repeats of both the Mizm1 and Mizm2 motifs upstream of the transcription start site, pGL3e-Mizm1 containing four repeats of the P1 probe sequence, and pGL3e-Mizm2 containing four repeats of the P2 probe sequence. (B) Miz-1 overexpression in HeLa cells produces a statistically significant increase in luciferase reporter activity with all of the three reporter vectors containing putative Miz-1 binding motifs. (C) Three mutant luciferase reporter vectors were constructed, containing two (Mizm1mut2), three (Mizm1mut3), or five (Mizm1mut5) changes in highly conserved bases of the motif. (D) Miz-1 overexpression produces a statistically significant increase in luciferase reporter activation in the presence of Mizm1, but the effect is eliminated by mutating as few as two bases in the motif. (E) Overexpression of c-Myc does not synergize with Miz-1; instead, c-Myc overexpression produces a statistically significant increase in luciferase activity for all conditions: with or without Miz-1 overexpression, and with or without the presence of Miz-1 binding motifs. Luciferase expression was normalized to expression of the Renilla luciferase control reporter vector and to luciferase expression in untreated HeLa cells. * p<0.05; ** p<0.01; *** p<0.001. EV  =  empty vector control; RC  =  reverse complement.