Figure 5.

Effects of TNF-α on HSD17B2 and HSD17B1 expression in endometrial cells. Endometrial cells were treated with increased concentrations of TNF-α. A, Immunoblotting for HSD17B2. HSD17B2 protein levels were determined 72 h after treatment with different concentrations of TNF-α (1, 2, or 5 ng/ml). B, Bar graph of densitometry analysis of immunoblots. Intensities of the bands are expressed as multiples of protein levels in control cells. The mean values ± sem of three separate immunoblots. C, Real-time RT-PCR analyses of HSD17B2 transcript. The cells were treated with TNF-α (1, 2, or 5 ng ml⁻¹) for 12 h followed by total RNA isolation from the cells, and the HSD17B2 mRNA expression was determined by real-time RT-PCR. The threshold cycle value HSD17B2 gene was normalized to the threshold cycle value of GAPDH. D, HSD17B2 reporter gene assays. The cells were stably transfected with pGL3 HSD17B2 reporter, which contains the human HSD17B2 promoter from −1588 to +165 bp, and treated with TNF-α (1, 2, or 5 ng ml⁻¹) for 48 h. D, Real-time RT-PCR analyses of HSD17B1 transcript in EM1 treated with TNF-α (1, 2, or 5 ng ml⁻¹) for 12 h followed by total RNA isolation from the cells. The HSD17B1 mRNA expression was determined by real-time RT-PCR. The threshold cycle value HSD17B1gene was normalized to the threshold cycle value of GAPDH. The results are expressed as mean ± sem of independent experiments.