Figure 5.

Vitamin D up-regulation of UPP1 expression and suppression of uridine induced DNA damage in human colonic organoids. Human colon organoids from 10 individuals were treated with 1α,25(OH)₂D₃ or ethanol, and expression of UPP1 was examined. (A) A 5.32-fold up-regulation of UPP1 mRNA at 6 hours after vitamin D treatment (P < .0005). (B) Representative immunofluorescence images of organoids treated with 1α,25(OH)₂D₃ and ethanol. The columns are nuclear DAPI staining (blue), Upp1 staining (green), and the overlay of DAPI and Upp1. Localization of Upp1 protein was observed in both cytosol and nucleus. Scale bar indicates 64 µm. (C) Quantitated mean gray values of Upp1 expression in 1α,25(OH)₂D₃-treated cells that were normalized to ethanol and showed a 3.42-fold up-regulation of Upp1 protein expression after 24 hours of vitamin D treatment (P = .0020). (D) Representative FLARE images of human colonic organoids treated with uridine plus ethanol and uridine plus vitamin D. The arrowhead points to the tail representing DNA damage. (E) DNA damage as estimated by tail moment using ImageJ is shown. Representative comparisons and P values are shown on the graph for clarity. There was significantly increased tail moment with uridine alone and with uridine plus ethanol treatments compared with control (both P < .0001). There was significantly less tail moment in cells treated with the combination of uridine plus vitamin D compared with cells treated with uridine plus ethanol or with uridine alone (both P < .0001). DAPI, 4′,6-diamidino-2-phenylindole.