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. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: J Steroid Biochem Mol Biol. 2017 Jun 19;172:117–129. doi: 10.1016/j.jsbmb.2017.06.010

Figure 3. Effect of 1,25D Concentration on SIRT1 Enhancement of VDRE-mediated Transcription in HEK293 Cells.

Figure 3

HEK293 cells were transfected with the XDR3 VDRE-reporter plasmid (250 ng/well) and 5 ng pCMV-empty (-SIRT1) or 5 ng pCMV-SIRT1 (+SIRT1). After 24 hours of transfection, the cells were refreshed with complete medium containing ETOH vehicle or the following final concentrations of 1,25D equal to: (A) 10 nM, (B) 1.0 nM, and (C) 0.1 nM. After an additional 22–24 hours, the cells were lysed and analyzed sequentially for Firefly and Renilla luciferase activity as described in Methods. The Firefly/Renilla ratio was then multiplied by a scaling factor (usually 10,000), and converted to fold-effect of SIRT1 compared to cells treated with 10 nM 1,25D and transfected with empty vector control, which was set at 1.0-fold in (A) for standardization and comparison of (B) and (C) to (A), and to streamline data presentation. The fold-effect average for six biological replicates (n=6) was calculated for each experimental treatment group and standard deviation values were computed and expressed as error bars. P-values are noted in the figure panels.