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. 2018 Feb 22;10(2):247. doi: 10.3390/nu10020247

Figure 4.

Figure 4

Effects of OA on CYP27B1 and CYP24A1 mRNA, protein expressions, and promoter activities in HKC-8 cells. The expression levels of (A) CYP27B1 mRNA; (B) CYP24A1 mRNA; (C) CYP27B1 protein; (D) CYP24A1 protein as well as the promoter activities of (E) CYP27B1 and (F) CYP24A1 in HKC-8 cells were studied. HKC-8 cells were treated with vehicle (0.1% ethanol), 10−7 M PTH (1–34, human), 10−5 M Foskolin or 10−8 M 1,25(OH)2D3, and 10−9 M–10−5 M OA for 24 h. Cells were harvested by Trizol reagent at indicated time for RT-PCR and real-time PCR analysis (A,B). Relative gene expression was normalized by GAPDH. Total protein was extracted by lysis buffer and separated by SDS-PAGE and immunoblotted with anti-CYP27B1 (C,E), anti-CYP24A1 (D,E) antibody and normalized with β-actin expression. Promoter activities (F,G) were measured by dual luciferase assay and data were normalized against a thymidine kinase (TK) reporter construct. Results are presented as mean ± SEM (n = 3) and analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the control.