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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 1. BAG-1 negatively regulates TGFB1 expression.

Figure 1

(A) Western blot analysis confirmed that BAG-1 protein levels are reduced by BAG1 siRNA compared to control siRNA in HCT116 and SW480 colorectal carcinoma cells. Q-RT-PCR (B) and ELISA (C) were used to assess the effects of depleting BAG-1 expression on TGFB1 mRNA and TGF-β1 protein levels in HCT116 and SW480 cells. (C) Conditioned medium was collected from approximately 70% confluent cultures in serum-free medium containing 0.1% BSA. 24h after siRNA transfection, cell culture supernatants were collected following a further 24 hour incubation, centrifuged to remove debris and the amount of TGF-β1 was measured by ELISA, and normalised to cell counts from a parallel flask. Data points show the mean of three independent experiments ±SD bars, * p≤0.05; ** p≤0.01; *** p≤0.001 (D) HCT116 cells stably transfected with either the empty vector pBIG2i (HCT116/BIG2i) or pBIG2i/BAG-1L expression construct (HCT116/BAG-1L) were incubated with or without 2μg/mL Doxycycline (Dox) for 24 hours then BAG-1 protein levels were determined by immunoblot; equal loading was confirmed by α-tubulin. Note the BAG-1L expression above endogenous levels in the pBIG2i/BAG-1L transfected cells is due to the inherent leakiness of the Dox inducible system. (E) HCT116 cells stably expressing either the vector only (BIG2i) or the inducible BAG-1L construct (BIG/BAG-1L) were treated for 24 hours with 2μg/mL Dox. BAG1 and TGFB1 mRNA expression levels were then assessed by Q-RT-PCR and presented as the fold-change in mRNA abundance in Dox-treated cells relative to the non-Dox-treated controls. Data points show the mean of three independent experiments ±SD, ** p≤0.01.