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. Author manuscript; available in PMC: 2013 Sep 25.
Published in final edited form as: Mol Cell Endocrinol. 2012 Mar 28;361(1-2):31–39. doi: 10.1016/j.mce.2012.03.008

Figure 5. Effect of Sp3 or Sp1 knockdown on 1,25(OH)2D3-induced and PMA-enhanced CYP24A1 promoter activity.

Figure 5

Proliferating Caco-2 cells were transfected with one of three siRNAs: scrambled ((−)siRNA), Sp1, or Sp3. (A) PCR analysis of Sp1, Sp3 and VDR mRNA expression 48 h after siRNA transfection. Data were normalized to the expression level observed in the scrambled siRNA group (mean±sem, n=4). * = significantly different than the other groups within a gene group (p < 0.05). (B) Western blot analysis on whole cell extracts 72 h after Sp3 siRNA transfection. Samples were analyzed for Sp3, VDR or beta actin protein levels. Four Sp3 isoforms were observed: L1, L2, M1 and M2. (C) CYP24A1 reporter gene activity. 48 h after siRNA treatment cells were transfected with a CYP24A1 promoter-luciferase reporter gene. 15 h later cells were preincubated with PMA (100 nM, 10 min), followed by treatment with 1,25(OH)2D3 or ethanol vehicle (10 nM, 4 h in the absence of PMA). Reporter gene data were expressed relative to the 1,25(OH)2D3-induced expression of the reporter gene (calculated as 1,25(OH)2D3/vehicle = 1) (mean±sem, n=4). The values with different letter superscripts are significantly different from one another (p < 0.05, Fisher’s protected LSD). Data in each panel are representative of three independent experiments.