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. 2014 Dec 12;16:496. doi: 10.1186/s13058-014-0496-5

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© Guturi et al.; licensee BioMed Central. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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β-catenin/TCF4 as well as c-Myc regulates p68 promoter. (a) Schematic representation of human and mouse p68 promoters. (b) Activity of human p68 promoter (pGL3-hp68) was determined by luciferase assay in HEK293T, MCF7 and HCT116 cells after 36 h of transfection. Values represent the firefly luciferase activities normalised to renilla luciferase activities. (c) Similarly, pGL3-hp68 activity was determined in MCF7 cells treated with 20 mM lithium chloride (LiCl) for 2 h and 8 h. (d) Luciferase activities were determined in MCF7 cells transfected with human wild-type pGL3-hp68, or mutated transcription factor 4 (TCF4) constructs like pGL3-hp68-M1, pGL3-hp68-M2, pGL3-hp68-M3 and pGL3-hp68-M4 independently, along with pEGFP-β-catenin. (e) Mouse p68 promoter (pGL3-mp68) activities were determined in MCF7 cells transfected with either wild-type pGL3-mp68 or mutated TCF4 sites containing constructs pGL3-mp68-M1 and pGL3-mp68-M2 independently along with pEGFP-β-catenin. (f) Cross-linked chromatin fragments of MCF7, MDA-MB 231 and HCT116 (human) as well as 4T1 (mouse) cells were immunoprecipitated with respective antibodies as depicted in the figure. DNAs were isolated and PCR-amplified using primer sets designed from the promoter regions of p68 and cyclin D1 genes. Densitometry values are given below the images. (g) MCF7 cells were treated with either Wnt3a-CM or empty vector (EV)-CM (control). DNAs were isolated from cross-linked chromatins immunoprecipitated with the indicated antibodies and PCR-amplified using primer sets designed from the promoter regions of p68 and RPL30 genes. Densitometry values are given below the images. (h) β-catenin was knocked down in HCT116 and 4T1 cells using small interfering RNA (siRNA). Control siRNA (si ctrl)-transfected cells were kept as control. DNA fragments were isolated from cross-linked chromatins immunoprecipitated with the indicated antibodies and PCR-amplified using primer sets designed from the promoter regions of p68 and cyclin D1 genes. Input: 2.5% (f and g) or 5% (h) of total DNA isolated from cross-linked chromatin without immunoprecipitation. Densitometry values are given below the images.