On targetplus non targeting control pool sirna

Calcitriol (1,25-(OH)₂ vitamin D₃) was purchased from Cayman Chemical (Cat #71820) and used at 100 nM unless otherwise noted. Radioimmunoprecipitation assay buffer (RIPA) (Cat #R0278), protease and phosphatase inhibitor cocktails (Cat #P8340, Cat #P5726), and cycloheximide (Cat #01810) were purchased from Sigma Aldrich. FBS was purchased from Seradigm (Cat# 97068-085). IL-2 was purchased from Miltenyi Biotec (Cat #130-097-743). Clarity enhanced chemiluminescence (ECL) reagent (Cat #170-5061) and PVDF membrane and filter paper (Cat #170-4274), were purchased from BioRad. RPMI 1640 (Cat #10-00-CV) and Pierce bicinchoninic acid (BCA) protein assay kit (Cat #PI23225) were purchased from ThermoFisher Scientific. Polyacrylamide gels (4–12%; Cat #NW04125BOX) were purchased from Life Technologies. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethozyphenyl)-2-(4-sulfonphenyl)-2H-tetrazolium (MTS) Cell Proliferation Colorimetric Assay Kit was purchased from BioVision (Cat #K300-2500). EB1089 was purchased from R&D Systems (Cat #3993). 25(OH)D₃ was purchased from Cayman Chemical (Cat #9000683). ON-TARGETplus Pooled Human VDR siRNA (Cat #L-003448-00-0005) and ON-TARGETplus Control Non-Targeting Pool siRNA (Cat #D-001810-01-05) were purchased from Dharmacon.

STAT6 siRNA knockdown utilized the Invitrogen Neon Transfection System (Cat #MPK10096). NKL cells were plated at 2.5 million cells/mL and treated with 50 or 100 nM SMARTpool ON-TARGETplus STAT6 siRNA (Dharmacon, Cat #L-006690-00-0005) or 50 nM ON-TARGETplus Control Non-Targeting Pool siRNA (Cat #D-001810-01-05) for 48 h. Protein was harvested to assess the knockdown status via western blot. Knockdown percent was calculated by quantifying and normalizing STAT6 to β-actin for all three conditions, then normalizing the 50 nM and 100 nM siRNA to the scramble siRNA. This ratio was subtracted from 1, then multiplied by 100, to give the percent knockdown.

For p53 and CK2α′ knock-down, STHdh cells were transfected with FlexiTube siRNA (5 nmol) from Qiagen using DharmaFECT1 per manufacturer’s guidelines. As a negative control, ON-TARGETplus control non-targeting pool siRNA (Dharmacon) was used. Cells were collected 24 h after transfection. RNA was extracted from cells and mouse striatal tissues by using the RNeasy extraction kit (Qiagen) according to the manufacturer’s instructions. cDNA was prepared using the Superscript First Strand Synthesis System (Invitrogen). SYBR green based PCR was performed with SYBR mix (Roche). The qPCR amplification was performed using the LightCycler 480 System (Roche). Each sample was tested in triplicate and normalized to GAPDH levels. Primer details are provided in the key resources table and in Table S2. For ectopic expression, cells were transfected using Lipofectamine LTX Reagent (Invitrogen) and following Invitrogen protocol instructions (Invitrogen protocols 25-0946W). Cells were transfected with 10 μg DNA of Q23-GFP (RRID:Addgene_40261) Q74-GFP (RRID:Addgene_40262), p53-FLAG (10838, RRID:Addgene_10838), and harvested 24-48h post-transfection. For cytosolic and nuclear fractionation, cells were fractionated using the NE-PER Extraction Kit (Thermofisher, 78833) as per manufacturer’s instructions. RT-qPCR data for all genes was normalized using GAPDH as control.