Journal: PLoS ONE
Article Title: Cyclic AMP Enhances TGF? Responses of Breast Cancer Cells by Upregulating TGF? Receptor I Expression
Figure Lengend Snippet: The forskolin effect on TβRI requires transcription to be active, but is independent of CREB. (A) MDA-MB-231 cells were incubated with either actinomycin to block transcription or mock-treated for 24 h and analyzed for TβRI RNA levels by Q-RT-PCR. (B-D) Cells were transfected with either siCREB, siLuc or no siRNA, incubated for three days, treated for an additional 3 or 24 h with forskolin or mock (D) and analyzed for TβRI RNA (B, D) or protein levels (C) by Q-RT-PCR or by the Western blot technique (ns = non-specific band), respectively. (E) Cells were transfected with siSix1 (S) or siLuc (L), incubated for three days, treated with forskolin or mock o/n and analyzed for Six1 and TβRI RNA expression by Q-RT-PCR. (F) The TβRI promoter is responsive to cAMP. MDA-MB-231 cells were transfected with TβRI promoter/dual luciferase construct, incubated o/n and treated with forskolin (FSK) or mock for 6, 18 or 24 hours as indicated and analyzed for firefly and renilla (control) luciferase. (G) MDA-MB-231 cells were incubated with forskolin, HDACi III, forskolin plus HDACi III or mock-treated for 24 h and analyzed for TβRI expression by Q-RT-PCR. Each bar represents the mean value ± S.D. of 3 independent experiments. Relative promoter activity denotes the ratio of firefly to renilla luciferase activity. Each bar represents the mean value ± S.D. of 3–10 independent experiments. * p-value
Article Snippet: Correct sequence and positioning of the TβRI promoter fragment within the pGL4 vector were confirmed by DNA sequencing (MWG Eurofins).
Techniques: Multiple Displacement Amplification, Incubation, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, RNA Expression, Luciferase, Construct, Expressing, Activity Assay