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. 2021 Jan 5;24(1):101974. doi: 10.1016/j.isci.2020.101974

Figure 4.

Figure 4

1,25D inhibits Dickkopf family member 3 (DKK3)

(A) RT-qPCR of DKK3 expression in prostate cell lines (LAPC4, PC3, RWPE1, 957E), monolayer prostate epithelial cells (PrE 2D), and organoid PrE cells (3D) grown in vehicle or 10 nM 1,25D. Monolayer cells were treated for 24–72 h, and organoids were treated for 2–3.5 weeks. Relative quantitation is normalized to HPRT1; error bars represent standard deviation of technical RT-qPCR pipetting replicates. Cells from multiple sources represent biological replicates. p value represents interaction outcome of grouped two-way ANOVA comparison of vehicle versus 1,25D in PrE cells using different patients as biological replicates; individual p values are shown (∗p < 0.1; ∗∗p < 0.05; ∗∗∗∗p < 0.001).

(B) Immunostaining for DKK3 (green) in a whole-mounted day 17 AA3 organoid counterstained with DAPI (blue) and phalloidin/F-actin (pink). Scale bar, 50 μm.

(C) ELISA quantification of secreted DKK3 in media collected from monolayer PrE cells grown in vehicle or 10 nM 1,25D for 72 h, normalized to the number of cells. Error bars represent the standard error of triplicate ELISA technical samples. Cells from multiple sources present biological replicates. p value represents interaction outcome of grouped two-way ANOVA comparison of vehicle versus 1,25D using different patients as biological replicates, individual p values are shown (∗∗p < 0.05; ∗∗∗∗p < 0.001).

(D) Western blot of DKK3 expression in cell lines and in monolayer and organoid PrE cells grown in vehicle or 10 nM 1,25D (sDKK3, secreted DKK3 variants; DKK3b, intracellular DKK3 variant); molecular weight marker shown (M.W.).

(E) VDR-ChIP sequencing peak near DKK3 in PrE cells treated with 1,25D for 3 h; IGV track showing 1,25D-treated minus vehicle-treated coverage (data from NCBI GEO accession number GSE124576).

(F) Western blot of monolayer AA1 prostate epithelial (PrE) cells transduced with control virus (siGFP) or siDKK3 virus and grown in vehicle, 10 nM 1,25D, or 50 ng/mL rDKK3 (upper left); molecular weight marker shown (M.W.). Representative images of siGFP- or siDKK3-treated AA1 PrE organoids grown in vehicle or 10 nM 1,25D for 2 weeks (lower left), with quantification of relative area (right). Area was normalized to the mean of the vehicle, showing ≥3 replicate wells for each condition; each dot represents an organoid. (∗p < 0.1;∗∗∗p < 0.01; ∗∗∗∗p < 0.001. Scale bar, 200 μm).

(G) Relative area of PrE organoids grown in vehicle or 10 nM 1,25D combined with 50 ng/mL rDKK3 treatment for 2 weeks. Area was normalized to the mean of the vehicle, showing ≥3 replicate wells for each condition, each dot represents an organoid. (∗∗p < 0.05; ∗∗∗p < 0.01; ∗∗∗∗p < 0.001).