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. 2017 Oct 19;7:13575. doi: 10.1038/s41598-017-14148-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The active promoter region of the MPP-2 gene contains the binding site for AP-2α that interacts with TFPI-2. (A) Western blots show the expression of TFPI-2 and dNLS-TFPI-2 in MDA231 stable cell lines. GAPDH was used as a loading control. (B) Representative immunofluorescent assays indicate that deletion of the NLS blocked the nuclear localization of TFPI-2 in dNLS-TFPI-2 cell line. Scale bars: 10 μm. An average of 100 cells of each cell type was examined. **P < 0.01 as determined by Student’s t-test. (C) Analyses of the effect of nuclear localized TFPI-2 on MMP-2 promoter activity. Luciferase reporter driven by the -1030 to -1 bp of the MMP-2 promoter and an internal control Renilla luciferase plasmid were co-transfected into stable MDA231 cell lines. The firefly luciferase activity is normalized to the Renilla luciferase activity and the relative luciferase activity is represented. The results are the average of a total of three independent experiments each carried out in triplicate. *P < 0.05, **P < 0.01 as determined by Student’s t-test and by one-way ANOVA followed by Tukey’s multiple comparison tests. (D) Analyses of active regions of the MMP-2 promoter. Left: schematic representation of the luciferase reporter constructs driven by 5′ nested deletions of the MMP-2 promoter. Relative positions of the promoter region in each construct are marked. The arrow indicates the putative transcription initiation sites for the MMP-2 gene. The green block indicates the potential binding element for AP-2, SP1 and PEA3. Right: MMP-2 promoter constructs and an internal control Renilla luciferase plasmid were co-transfected into cultured MDA231/TFPI-2 cells. The firefly luciferase activity from each construct was normalized to the Renilla luciferase activity and the relative luciferase activity is represented as a percentage of the MMP-2 promoter construct (-1030/-1). The results are the average of three independent experiments each carried out in triplicate, ± SD. P < 0.01 as determined by one-way ANOVA followed by Tukey’s multiple comparison tests.