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. 2016 Nov 25;6:37808. doi: 10.1038/srep37808

Figure 6. Transcriptional responses from the estrogen responsive CXXC5 region-driven gene reporter.

Figure 6

(a) MCF7 cells grown in medium containing CD-FBS for 48 h were transiently transfected with pGL3 bearing none (Basic-Luc), the estrogen responsive region of CXXC5 (CXXC5-Luc) or OXT (OXT-Luc) driving Firefly Luciferase cDNA expression as the reporter in the absence (EtOH, 0.01%) or presence of 10−9 M E2 for 24 h. The transfection efficiency was monitored by the co-expression of pCMV–RL that drives the expression of Renilla Luciferase cDNA. 24 h later, cellular extracts were subjected to luciferase assays. Shown is the mean ± SD of three independent experiments performed in triplicate. Firefly/Renilla luciferase activities are presented as fold change compared to EtOH control of pGL3-Basic, which is set to 1. *a and *b indicate significant difference from E2 of Basic-Luc and the corresponding EtOH control, respectively. (b) MCF7 cells transfected with CXXC5-Luc treated without (EtOH, 0.01%) or with 10−9 M E2 and/or 10−7 M ICI for 24 h were subjected to luciferase assays. Shown is the mean ± SD of three independent experiments performed in triplicate. Firefly/Renilla luciferase activities are presented as fold changes compared to EtOH, which was set to 1. (c) MDAMB231 cells were transfected as described in (A) with Basic-Luc, CXXC5-Luc, or OXT-Luc reporter together with pCDNA-Flag-ERα vector. Cells were also co-transfected with pCMV-RL for monitoring transfection efficiency. Results are the mean ± SD of three independent experiments performed in triplicate. Firefly/Renilla luciferase activities are presented as fold changes compared to EtOH of pGL3-Basic, which is set to 1. *a indicate significant change from EtOH of Basic-Luc; while *b denotes significant change of E2 compared to EtOH of CXXC5-Luc or OXT-Luc. (d) MDAMB231 cells were transfected with CXXC5-Luc or mutCXXC5-Luc vector, the latter which bears a mutant sequence that changes the ERE sequence in CXXC5 to a non-ERE, together with pcDNA-Flag-ERα vector. Cells were treated without (EtOH, 0.01%) or with 10−9 M E2 for 24 h. Shown is the mean ± SD of three independent experiments performed in triplicate. The normalized Firefly/Renilla luciferase activities are presented as fold change compared to EtOH of CXXC5-Luc, which was set to 1.