ABSTRACT: WM164, a human melanoma line, was a gift from Dr M. Herlyn (Wistar Institute, Philadelphia, PA, USA). The cells were cultured using DMEM media supplemented with 10% serum (fetal bovine serum while growing cells and charcoal-treated serum during incubation with vitamin D compounds), 1% antibiotics and 5 g/mL insulin (Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in 75 cm2 plastic bottles (TPP–Techno Plastic Products AG, Trasadingen, Switzerland) with a filter in an incubator at 37 C with 5% CO2. CRISPR/Cas Knock out of the VDR. The clustered regularly interspaced short palindromic repeats (CRISPR) genetic engineering method was carried out to knock out VDR expression in the WM164 cell line as described in (Cancers 2021, 13, 3111. https://doi.org/10.3390/cancers13133111). The resulting cells with the VDR gene knocked out are designated as WM164 VDR KO, and the cells transduced with an “empty” lentivirus served as a control (WM164 scramble). VDR expression was checked for scramble and VDR KO lines before experiments were performed and further every 4 months after lentivirus treatment to ensure that VDR was not expressed in the WM164 KO. Sources of Vitamin D3 Compounds: Both 1,25(OH)2D3 and 25(OH)D3 were purchased from Sigma. 1,20(OH)2D3 and 20(OH)D3 were chemically synthesized. RNA SEQ: VDR-KO or scramble WM164 cells were plated onto 100 mm2 Petri dishes and treated with 1,25(OH)2D3, 25(OH)D3, 20(OH)D3, 1,20(OH)2D3 or ethanol (control) in triplicates. Cells were incubated in 10% charcoal-treated serum for 24 h. Cells were further harvested and RNA isolated using Absolutly RNA Miniprep Kit (Stratagen) . Approximately 120 ng/ul of each sample was sent for RNAseq analysis. BGI@CHOP genomics company sequenced the samples on Illumina HiSeq TM, analyzed, and sent us the results.
