FIGURE 3.

KLF16 directly repressed CYP1A1 expression in primary endometrial cells. a, primary endometrial cells were transfected with KLF16 siRNA, which resulted in >90% diminished protein levels as detected by Western blot using anti-KLF16. b, 249-bp amplicon of the KLF16 promoter showing the location of the forward and reverse primers (underlined bases) as well as the BTE (boldface type) located at base pairs −55 to −41 relative to the transcription start site. c, ChIP was performed for endogenous KLF16, which confirms that KLF16 binds the CYP1A1 promoter BTE-element in primary endometrial cells. d, real time PCR was used to determine the effect of decreased KLF16 expression on CYP1A1 mRNA expression in these cells. CYP1A1 normalized to a housekeeping gene (β₂-microglobulin) was increased 2-fold (p < 0.05) in cells transfected with KLF16 siRNA compared with control, which is similar in magnitude to our findings in the uterine cell line. e, primary uterine cells were transfected with FLAG-KLF16 or corresponding empty vector along with the 6×CYP1A1-BTE luciferase construct. Results of luciferase-reporter assays normalized to protein expression revealed that KLF16 significantly repressed luciferase activity compared with empty vector control (50%; *, p = 0.01).