Fig 2: Uterine and placentomal TRPV6 and CaBP-9k mRNA expression. TRPV6 and CaBP-9k mRNA expression in the intercaruncular wall (Figure 1A, C) and in the placenta (n = 3 animals/month) (Figure 1B, D) from the 2nd to 9th months of pregnancy, determined by real-time PCR. Bars with ** differ significantly (p < 0.05) compared to the second month (*). Data were analysed using a parametric one-way analysis of variance (ANOVA), followed by Dunnett’s comparison test and are presented as means ± SD. The nonparametric dataset of placental CaBP-9k was analysed using the Kruskal-Wallis test, followed by Dunn`s post test.

Fig 3: Tv1 co-localizes with TRP Channels. (a) Intensities of TRPV6 and TRPC6 channel expressions are compared for 1MEA and BNL.CL2 cells at 800nm channels after normalizing at 700 nm channel for cell-tag. TRPV6 and TRPC6 in 1MEA have significantly increased levels (p < 0.05) of cells compared to BNL cells. (b) Co-localization analysis of Tv1 peptide labeled with fluorescein dye (Tv1-Fam) with TRPV6 and TRPC6 channels in 1MEA cells using MOC (manders overlap coefficient). Y-axis indicates incubation of specific channel, TRPV6 or TRPC6. The X-axis indicates the filters used for imaging, from left to right: DAPI, GFP, DAPI & CY5, DAPI, GFP, and Cy5. High MOC values (0.80 ± 0.01 and 0.9 ± 0.05 for TRPC6 and TRPV6, respectively) confirmed Tv1 co-localizes with these channels. (c) A specific blocker for TRPV6 channel, truncated SorC labeled with rhodamine (TruncSorC-Rho), was used to demonstrate colocalization as an indicator of activity. Both TruncSorC-Rho of Tv1-Fam with TRPV6 channels in its presence. High MOC for TruncSorC-Rho (0.88 ± 0.04) and Tv1-Fam (0.9 ± 0.05) with the channels indicate colocalization. Fluorescein labeled non-specific peptide ACP-Fam, probed with channel antibodies (TRPC6 and TRPV6) was used to examine non-specific green fluorescence and no colocalization was detected. Dyes and channels used: DAPI-staining nucleus for the presence of cells, GFP- green channel for the presence Tv1-Fam, Rho channel for the presence of Rhodamine labeled TruncSorC-Rho peptide, Cy-5 channel for the presence of ion channels as secondary antibodies used were cy-5 labeled.

Fig 4: Quantification of DAB+ cells along the ES reveals staining gradients along the ES epithelium increasing from the iES to the eES. The y-axis shows the percentage of DAB+ cells relative to all ES epithelial cells. For cell counting, both the iES and eES were each divided into ten equal segments. The ticks on the x-axis correspond to each of the ten consecutive segments of the iES (left x-axis) and eES (right x-axis). The first two segments represent the endolymphatic duct epithelium. The values are the percentages of the total number of cells in the respective ES segment and are given as mean values ± SD from six animals. a Quantification of DAB+ cells for CaSR and the apical Ca2+ channels TRPV5 and TRPV6. b Quantification of DAB+ cells for the cytoplasmatic proteins SERCA1 and SERCA2. c Quantification of DAB+ cells for proteins located in the basolateral membrane

Fig 5: Real-time PCR. VDR, CB, and TRPV6 mRNA expression levels in the different intestinal segments in the horse intestine (DD = duodenum, JE = jejunum, IL = ileum, CC = cecum, CAV = colon ascendens ventrale, CAD = colon ascendens dorsale, CD = colon descendens) were determined by real-time (TaqMan) PCR. Expression was normalised to the expression of ß-actin. VDR mRNA was found in each intestinal segment, but the amount and distribution patterns showed distinct inter-individual variations between the animals investigated. There were no significant differences between VDR mRNA expression levels in the small and large intestine (p > 0.05) (Figure 2A). The duodenum expressed the highest levels of TRPV6 and CB mRNAs (p < 0.05) (Figure 2B, C).