Figure 2. SIRT1 Enhances VDRE-mediated Transcription in Vitamin D Target Cells.

(A) HEK293 cells were transfected with 5 ng pCMV-empty (-SIRT1) or 5 ng pCMV-SIRT1 (+SIRT1) in addition to 250 ng of XDR3 VDRE-luciferase containing two copies of the distal DR3 VDRE from the human CYP3A4 gene [56]. Twenty-four hours post-transfection, the cells were exposed to fresh medium containing ethanol (the vehicle control, ETOH) or 1 nM 1,25D in ethanol. 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 1,25D-treated cells transfected with empty vector control, which was set at 1.0-fold for standardization 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. (B) A similar experiment as in (A) was conducted with the PER6 VDRE-luciferase construct; however, the fold-effect average for twelve biological replicates (n=12) was calculated for each experimental treatment group. (C) A similar experiment as in (A) was conducted using TE85 osteoblast-like cells instead of HEK293 cells. P-values are noted in the figure panels.