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. Author manuscript; available in PMC: 2011 Jan 1.
Published in final edited form as: Int J Cancer. 2010 Jan 1;126(1):81–89. doi: 10.1002/ijc.24696

Figure 3. KLF5 inhibits E2/ERα-induced promoter activity and expression of both c-Myc and Cathepsin D (CSTD) in ER-positive breast cancer cells, apparently by reducing the binding of ER to c-Myc and CSTD promoters.

Figure 3

(A) Expression of KLF5 inhibits estrogen-promoted ER responsive promoter-mediated luciferase reporter activity in MCF-7 cells. E2 treatment was at 1 nM for 20 hours. Asterisks indicate statistically significant differences in luciferase activities between cells transfected with pcDNA3 and cells transfected with pcDNA3-KLF5 in the presence of estrogen (p < 0.05). (B, C) Stimulation of c-Myc expression by estrogen is suppressed by expression of KLF5 in MCF-7 (B) and T-47D (C) cells, as detected by real-time PCR. Cells were transfected with pcDNA3 or pcDNA3-KLF5, and 30 hours later were treated with 100 nM E2. (D) Binding of ERα to c-Myc promoter is reduced by KLF5, as detected by chromatin immunoprecipitation (ChIP) assay combined with regular PCR (left) and real-time PCR (right) assays in MCF-7 cells. MCF-7 cells transfected with pcDNA3-KLF5 or pcDNA3.1 were incubated in the presence or absence of 25 nM E2 for 1 hour, and antibody against ERα was used to precipitate DNA-protein complexes. Precipitated DNA was analyzed by PCR with primers specific to Myc promoter. (E, F) Measurement of Cathepsin D (CSTD) mRNA levels by real time PCR in MCF-7 (E) and T-47D (F) cells transfected with KLF5 or vector control (pcDNA3). Cells were treated with 100 nM E2. In panels B, C, E and F, asterisks indicate statistically significant differences in mRNA expression between cells transfected with pcDNA3 and cells transfected with pcDNA3-KLF5 in the presence of estrogen (P < 0.05). (G) Detection of ERα binding to CSTD promoter by ChIP assay combined with regular PCR (left) or real-time PCR (right) assays in MCF-7 cells transfected with KLF5 and vector control. The value shown in panels D and G was normalized with that of inputs, with that for the group of KLF5 plus E2 treatment defined to 1. These experiments were repeated twice and consistent results were obtained.