Figure 5. Expression of Vitamin D₃ signaling components in individual microenvironments of the mammary gland.

A) Real time PCR 1α-OHase (a) and vitamin D₃ receptor (VDR) (b) in total mammary gland (Mammary Gland), purified epithelial organoids (Organoids), and cleared mammary fat pad (Mammary Adipose). Data are expressed relative to 18S RNA (normalized gene equivalence) and represent mean s.e.m. of triplicate runs. Similar letters above the bars designate comparison and significance in the expression of 1α-OHase or VDR within total mammary glands or the individual microenvironments of the mammary gland compared to a traditional target tissue for 1α-OHase and VDR expression - p<0.05. B) Western blot of VDR,1α-OHase, Perilipin, Cytokeratin 18 (cyto 18), and α-tubulin in total mammary gland (MG), cleared mammary fat pad (MG fat only), purified epithelial organoids from wild type (WT-E) and VDR knockout (KO-E) mice, and kidney (Kid). C. Bioactivation and signaling through the VDR to regulate 24-OHase in VDR WT and KO primary preadipocytes. 24- OHase induction by 25D₃ and 1,25D₃ in VDR WT primary mammary preadipocytes compared to VDR KO that lack a functional VDR. 24-OHase mRNA expression normalized to 18S from cultured cells treated for 48h with ethanol (Et), 25D₃, or 1,25D₃. *- p <0.05 compared to vehicle control at each genotype.