Supplementary Materials Supplemental Data supp_26_9_3703_v2_index. Sp1 AZD-9291 distributor and Sp3 (EC503107 M), transcription factors known to increase DNMT3a expression, and inhibition of these transcription factors abrogated the PGE2 increase of DNMT3a expression. These findings were specific to fibroblasts, as PGE2 decreased DNMT1 and DNMT3a expression in RAW macrophages. Taken together, these findings establish that DNA methylation is usually regulated by a ubiquitous bioactive endogenous mediator. Given that PGE2 biosynthesis is usually modulated by environmental toxins, various disease says, and commonly used pharmacological brokers, these findings uncover a novel mechanism by which alterations in DNA methylation patterns may arise in association with disease and certain environmental exposures.Huang, S. K., Scruggs, A. M., Donaghy, J., McEachin, R. C., Fisher, A. S., Richardson, B. C., Peters-Golden, M. Prostaglandin E2 increases fibroblast gene-specific and global DNA methylation increased DNA methyltransferase expression. alterations in specific second messengers (DNMT. MATERIALS AND METHODS Cell culture Primary fetal (IMR-90) and adult (CCL-210, CCL-204, and CCL-209) lung fibroblasts were purchased from American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) GTF2H and 1% penicillin/streptomycin. Cells from the murine monocyte-macrophage leukemic cell line, RAW 264.7, were cultured in Roswell Park Memorial Institute medium. All experiments were performed on cells at passages 5C9. Cells were plated unless otherwise noted at 5 105 cells/well in 6-well AZD-9291 distributor plates, serum starved overnight, and treated with the following reagents: PGE2 (10?9 to 10?6 M), the prostacyclin [prostaglandin I2 (PGI2)] analogs iloprost (1 M) and treprostinil (1 M), PGD2 (1 M), the thromboxane A2 analog U-4419 (1 M), and the EP2 agonist butaprost free acid (500 nM) (all from Cayman Chemical, Ann Arbor, MI, USA); aspirin (200 M), the DNA-damaging agent temozolomide (100 and 250 M), and actinomycin D (2.5 g/ml) (all from Sigma-Aldrich, St. Louis, MO, USA); IL-1 (10 ng/ml; BD Biosciences, Sparks, MD, USA); the EP3 agonist ONO-AE3-248 (100 nM), the EP4 agonist ONO-AE1-329 (100 nM), and the EP4 antagonist ONO-AE3-208 (100 nM) (kind gifts from Ono Pharmaceuticals, Osaka, Japan); the adenyl cyclase activator forskolin (100 M; EMD Chemicals, San Diego, CA, USA); the EP2 antagonist AH6809 (10 M; Enzo Life Sciences, Farmingdale, NY, USA); and the Sp1/Sp3 inhibitor mithramycin (25C50 nM; Tocris Bioscience, Bristol, UK). For small interfering RNA (siRNA) experiments, cells were cultured to 30C50% confluence and transfected with siRNAs against DNMT1, DNMT 3a, and DNMT 3b (Qiagen, Valencia, CA, USA) using Lipofectamine LTX (Invitrogen) for 48 h in OptiMEM medium (Invitrogen) before being treated with or without PGE2. Specificity of the siRNAs was confirmed by real-time AZD-9291 distributor RT-PCR and has been reported previously (9). Global and gene-specific DNA methylation Genomic DNA was isolated from 1 106 cells using DNeasy (Qiagen). Levels of global DNA methylation were assayed using the MethylFlash Methylated DNA Quantification Kit from Epigentek (Farmingdale, NY, USA) according to the manufacturer’s protocol. For gene- and site-specific analysis, 1 g of genomic DNA was subject to bisulfite conversion using the EZ DNA Methylation Kit from Zymo Research (Irvine, CA, USA). Bisulfite-converted DNA was analyzed for methylation at 27,578 CpG sites using the HumanMethylation27 BeadChip array (Illumina; San Diego, CA, USA) according to the manufacturer’s protocol. Signal intensity from methylated and unmethylated probes for all those sites was scanned around the Illumina BeadArray Reader and preprocessed using Illumina GenomeStudio software. The methylation status of individual CpG sites was verified by pyrosequencing. Bisulfite-modified DNA was amplified by PCR using biotin-labeled primers specific for the and promoter. The amplified product was then mixed with sequence-specific primers and analyzed for methylation at individual CpG sites using a Pyrosequencer (Qiagen). and amplification and sequencing-specific primers were obtained from EpigenDx (Worcester, MA, USA). Long interspersed element (LINE)-1 amplification and.