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. 2021 Jan 8;11(1):77. doi: 10.3390/brainsci11010077

Figure 6.

Figure 6

Analysis of Gli1/2 binding sites on zo-1 promotor by dual-luciferase reporter assays. (A) The sequence logo of the Gli1/2 binding motif. (B) Schematic of the 4 predicted Gli1/2 binding sites on zo-1 promotor and their binding sequences accordingly. The binding sites were located at −1392 to −1381 (site 1), −1318 to −1307 (site 2), −380 to −369 (site 3), and +32 to +43 (site 4) of the zo-1 promotor. (C,D) The zo-1 luciferase activities tested by applying a series of truncations (C) and site-targeted mutations (D) to the zo-1 promoter, along with pcDNA3.1-Gli1, pcDNA3.1-Gli2, and pRL-TK plasmids. The specific constructs used in the truncation assays (C) included pGL3-basic vector, pGL3-zo-1-promo-WT (containing promotor region from −1464 to +167), pGL3-zo-1-promo-truncation1 (from −1039 to +167), pGL3-zo-1-promo-truncation2 (from −499 to +167), pGL3-zo-1-promo-truncation3 (from −163 to +167), and pGL3-zo-1-promo-truncation2&mut3 (from −499 to +167 which lacking site 3). The specific constructs used in the site-mutation assays (D) included pGL3-basic vector, pGL3-zo-1-promo-WT (containing all 4 sites), pGL3-zo-1-promo-mut1 (lack of site 1), pGL3-zo-1-promo-mut2 (lack of site 2), pGL3-zo-1-promo-mut3 (lack of site 3), and pGL3-zo-1-promo-mut4 (lack of site 4). The luciferase activities were determined, and are presented as the ratios of firefly luciferase activity and renilla luciferase activity. The assays were performed with 3 replicates, and data are presented as mean ± SEM. ** p < 0.01. ns, no significance. (E) ChIP-qPCR validation of the Gli1/2 binding to the zo-1 promotor around −380 in hBMECs treated by TGFβ1 (50 ng/mL). ChIP was performed with anti-Gli2 antibody, and rabbit IgG was used as the negative control. ** p < 0.01. The assays were performed with at least 3 replicates, and data were presented as mean ± SEM.