Fig. 2.

UroA augments 1,25D-triggered transcriptional activation mediated by three different VDREs in cultured cells. (A) Chemical structure of urolithin A. (B) Effect of UroA on transcriptional activation driven by the XDR3 VDRE in HEK293 cells treated with 1,25D. Cells were exposed in culture to 10 nM 1,25D (added in EtOH vehicle) and either 10 μM, 15μM or 20 μM UroA (added in DMSO vehicle) for 24 h and transcription assessed by luciferase activity as described in Methods. Fold-effect over EtOH/DMSO vehicle control is listed at the top of each bar. Values are the average of three biological replicates (n = 3) ± Std. Dev. (C) Effect of UroA on transcriptional activation driven by the PER6 VDRE in HEK293 cells treated with 1,25D. Cells were exposed in culture to 0.5 nM 1,25D (added in EtOH vehicle) and 20 μM UroA (added in DMSO vehicle) for 24 h and transcription assessed by luciferase activity as described in Methods. Fold-effect of UroA over 1,25D control is listed at the top of the central pair of bars. Values are the average of six biological replicates (n = 6) ± Std. Dev. (D) Effect of UroA on transcriptional activation driven by the human CYP24A1 VDRE in HEK293 cells treated with 1,25D. The transfected VDRE-reporter was a plasmid consisting of a 5.5 kb natural promoter fragment of the human CYP24A1 gene fused to the luciferase reporter vector. Cells were exposed in culture 0.5 nM 1,25D (added in EtOH vehicle) and 20 μM UroA (added in DMSO vehicle) for 24 h and transcription assessed by luciferase activity as described in Methods. Fold-effect of UroA over 1,25D control is listed at the top of the central pair of bars. Values are the average of six biological replicates (n = 6) ± Std. Dev.