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. 2012 Nov 27;7(11):e50053. doi: 10.1371/journal.pone.0050053

Figure 6. ERα up-regulates the transcription of the MGARP promoter and acts in synergy with Sp1 to activate MGARP transcriptional activity.

Figure 6

A. pGL3-(−3 kb) reporter and different doses of ERα expression plasmid were co-transfected into HEK-293T cells to determine the dose-dependent manner of ERα in regulating the MGARP promoter by luciferase assay. B. The functional synergy between Sp1 and ERα was determined by cotransfection of the full-length MGARP promoter (−3 kb) or various promoter truncates with or without Sp1 plasmids for Luc assay as indicated. C. The synergystic transactivation activity of ERα and Sp1 under the stimulation of estrogens. The HEK-293T cells were treated with or without 10 nM of estradiol (E2) for 24 hours post transfection of pGL3-(−3 kb) and ERα. Subsequently, the Luc assay was performed at 72 hours post transfection. D. Knockdown of Sp1 diminishes the activation function of ERα on MGARP promoter. HEK-293T cells were co-transfected with pGL3-(−3 kb) reporter and ERα, together with Sp1-specific RNAi (630-RNAi or 1722-RNAi) or control RNAi, in the absence or presence of exogenous Sp1. *** represents p<0.001 and # represents p>0.05 (no significant difference). E. RT-PCR shows that down-regulation of Sp1 with Sp1-specific RNAi (630-RNAi or 1722-RNAi) results in a reduction in endogenous MGARP mRNA expression in HEK-293T cells when stimulated by 10 nM E2. The cells were treated with or without 10 nM E2 for 24 hours post transfection and total RNA was harvested at 72 hours post transfection for semiquantitative RT-PCR analysis.