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. Author manuscript; available in PMC: 2014 Mar 19.
Published in final edited form as: Oncogene. 2012 Oct 29;32(38):10.1038/onc.2012.480. doi: 10.1038/onc.2012.480

Figure 2. BAG-1 is a transcriptional regulator of TGFB1 gene expression.

Figure 2

(A) Schematic of the regulatory regions previously identified in the 5′ flanking region of the TGFB1 gene [adapted from 24]. (B) Luciferase activity of the TGFB1 reporter was assessed in HCT116 colorectal cancer cells transfected with either control siRNA or BAG1 siRNA, and treated with 1mM NaB to stimulate TGFB1 reporter activity (normalised to untransfected controls 100%). Depletion of BAG-1 had a small but non-significant effect on luciferase activity under basal conditions. However, on exposure to 1mM NaB for 24h, the luciferase activity of the +11/−1132 TGFB1 reporter was significantly increased in cells transfected with BAG1 siRNA when compared to control siRNA, which was not detected in cells treated with 10ng/mL TSA (an HDAC inhibitor). Data points show the mean of three independent experiments ±SD, ** p≤0.01. (C) Chromatin immunoprecipitation assays showed BAG-1 occupies the TGFB1 gene regulatory region between −323/−731 (−323/−453, −453/−560 and −560/−731) in HCT116 colorectal cancer cells. The GAPDH gene promoter was examined as a negative control for non-specific binding. Input DNA comprising 10% of total ChIP volume was used as a PCR control. Quantification represents BAG-1 binding as percent enrichment relative to input chromatin, IgG included as a negative control; mean of 3 independent measurements of a representative result, ±SD. Similar results were obtained in two independent experiments.