Fig 1: Regulation of endogenous OPG mRNA and protein by GATA-3.(A) wtGATA-3 and dnGATA-3 expression plasmids were transfected into HEK cells and the expression of GATA-3 and OPG mRNA was measured using qRT-PCR. (B) GATA-3 and scrambled shRNA constructs were transfected into HEK cells and GATA-3 and OPG mRNA expression after 48 h was measured using qRT-PCR. (C) wtGATA-3 and dnGATA-3 expression plasmids were transfected into HEK cells and the expression of GATA-3 and OPG proteins was detected using western blot analysis.(D) OPG secretion from HEK cells transfected with either the wtGATA-3 or dnGATA-3 plasmid for 48 h were measured in cell-conditioned culture medium using ELISA. *p < 0.05, **p < 0.01, n = 3 for qRT-PCR and western blot and n = 4 for ELISA. Full-length blots are presented in supplementary Figure 1.
Fig 2: Regulation of apoptosis by GATA-3 and OPG.(A) HEK cells expressing either a scrambled shRNA or a GATA-3 shRNA expressing plasmid were treated with etoposide (10 μM), and apoptosis was assayed by Western blot (left). HEK cells expressing a GATA-3 shRNA expressing plasmid were treated with purified OPG protein for 2 h before treatment with etoposide, and apoptosis was assayed by Western blot (right). Full-length blots are presented in supplementary Figure 1. (B) HEK cells expressing either a control plasmid or a GATA-3 shRNA expressing plasmid were treated with TNF-α (2 μg/mL) with or without pre-treatment with OPG for 2 h. Subsequent cell death was measured using TUNEL assays (left), and quantified with bar graphs (right). *p < 0.05, n = 3.
Fig 3: Transactivation of the OPG promoter by GATA-3.(A) A schematic diagram of two potential GATA-3 responsive elements on the human OPG promoter. The minimal OPG promoter was cloned from a HeLa genomic DNA library by PCR and cloned into the pGL-Luc vector. (B) Transactivation of the minimal OPG promoter by GATA-3 but neither RB-1 nor GTF2IRD1 using the dual luciferase assay. (C) Chromatin immunoprecipitation assay showing the binding of GATA-3 on the OPG promoter. The GFP-GATA-3 plasmid was transfected into HeLa cells and immunoprecipitated using an anti-GFP antibody. The PCR reaction showed that GFP-GATA-3 interacted with the chromatin containing the two potential GATA-3 responsive elements on the OPG promoter (from −895 to −461) but not the fragment outside the potential GATA-3 responsive element (from −2185 to −1873). (D) A dominant negative GATA-3 mutant lost its transactivation activity on the OPG promoter construct and could block the transactivation activity of the wild type GATA-3. **p < 0.01, ***p < 0.001, n = 5.
Fig 4: Identification of GATA-3 responsive elements on OPG promoter.(A) Luciferase results showed that mutations in either GATA-3 responsive element on the OPG promoter still supported GATA-3 transactivation while mutations in both elements blocked GATA-3 transactivation in HeLa cells after 24 hours of transfection. *p < 0.05, n = 5. (B) The gel shift assay showed that recombined GATA-3 could bind to the labeled G3RE1 and G3RE2 probes. This interaction could be blocked by a mutant G3RE1 or G3RE2 probe or excess non-labeled G3RE1 or G3RE2 probe. (C) The multiple minimal G3RE1 and G3RE2 could support GATA-3 transactivation. ** p < 0.01, n = 4.
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