Fig 2: VD supplementation could alleviate the epigenetics disorders on the VD metabolic genes and TGFβR1 by inhibiting the expressions of DNMT1(A and B) mRNA and protein expressions of hepatic VDR, VDBP, CYP27A1, CYP2R1, and CYP24A1.(C) mRNA expressions of renal VDR, CYP27B1, and CYP24A1.(D and E) Hepatic genome methylation and hydroxymethylation, respectively.(F) mRNA expression of mainly DNA methylating enzymes (DNMT1, DNMT3a, DNMT3b, TET1, TET2, and TET3).(G) Protein contents of DNMT1.(H–K) Methylation levels of VDR(H), CYP2R1 (I), CYP27A1 (J), and TGFβR1 (K), respectively, which were determined by bisulfite sequencing PCR (BSP), recorded through Chromas Software and estimated these results via C/(C + T). All pooled data were represented as mean ± standard deviation (n = 8/group). One-way analysis of variance was performed to compare the differences among these three groups, and then followed by the Student-Newman-Keuls test to determine the differences between each two groups. ∗Compared to the VDC group, p < 0.05. #Compared to the HFD group, p < 0.05. VDC, normal diet feeding group; HFD, high-fat diet feeding group; VD-HFD group, VD-supplemented HFD feeding group.

Fig 3: AF9 Is Required for Reconstituting an Activated State on the Promoter of Snail-Repressed Genes(A and B) ChIP assay was performed in MDA-MB-231-FLAG-AF9 OE (WT or Y78A) cells with the indicated antibodies. Primers against the promoter regions of CDH1, CLDN3, VDR, and CXADR were used to perform quantitative real-time PCR to measure the binding affinity of activated gene expression markers and histone acetyltransferases to these genes’ promoters. The relative binding affinities were normalized to the WT group with a FLAG antibody (A) or CBP antibody (B). Two-tailed Student’s t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant. Error bars represent the standard deviation (SD). (C and D) mRNA and protein levels of CDH1, CLDN3, VDR, and CXADR were tested in MDA-MB-231 and Hs578T cells with the indicated manipulations.

Fig 4: Human CYP24A1 as a direct vitamin D target gene in HaCaT cells(A) Robust induction of CYP24A1 by Vitamin D-mediated VDR. The wild-type (WT) HaCaT cells and VDR-KO HaCaT cells (VDR-KO) were treated with 1α,25(OH)2D3 for 8 h, and the extracted RNA was subjected to qRT-PCR. Data are expressed as the mean ± SE of three samples. (B) No VDR protein in the VDR-KO cells. Western blotting of VDR in the WT and VDR-KO cells. (C) The locus of human CYP24A1. Both VDRE-related motifs by JASPAR2020 and VDR-binding peaks registered in ChIP-atlas are shown.

Fig 5: E/QM status of PDAC dictates transcriptional response to VDR activation. (A) Schematic of experimental system in which tumor spheroids are grown in 3D culture conditions and dosed with 10 nM CalT or DMSO control on day 0 and day 2. Spheroids are harvested on day 5, and RNA is extracted for total RNA-seq. (B) CYP24A1 expression in a selection of PDAC cell-line spheroids following 5 d of CalT treatment compared with vehicle control as determined by RNA-seq, expressed as log10 reads per million (RPM). Error bars indicate SD. (C) Relative change in E/QM status of each PDAC cell line following 5 d of exposure to CalT expressed as fold change in metascore over vehicle-treated. (D) Expression heatmap of genes differentially expressed upon CalT treatment in all 6 PDAC cell lines. Data represents 2 to 3 independent experiments for each cell line. (E) Unsupervised clustering of RNA-seq data from 6 patient-derived PDAC cell lines at baseline (−) and after treatment with CalT (+). [Scale bar, log10(RPM+1).] (F and G) Expression heatmaps of differentially expressed genes following treatment of QM (F) and E (G) PDAC spheroids with CalT as determined by RNA-seq. Columns represent individual biological replicates. (H) Western blot demonstrating protein levels of FN1 and VDR in PDAC spheroids following treatment of PDAC spheroids with CalT for 5 d. (I) Enriched gene sets of interest from GSEA computing overlaps between CalT-induced genes in QM cell lines and hallmark, curated, and oncogenic signatures gene sets.