*2.9. Promoter Methylation of Inflammatory Genes*

*2.9. Promoter Methylation of Inflammatory Genes*  To assess changes in promoter methylation patterns in PF treated cells, a global DNA methylation array of genes involved in inflammation and autoimmunity was performed. The heat map in Figure S2A presents a range (from 0 to 100) of "M", the fraction of input genomic DNA containing 2+ methylated CpG sites in the targeted region of a gene. Genes that were shown to be impacted by DNA methylation by having significant *p*-values (<0.05) (Figure S2B) were C-C Motif Chemokine Ligand 25 (CCL25), Cluster of Differentiation 8a (CD8A), CCAAT Enhancer Binding Protein Beta (CEBPB), Dipeptidyl peptidase 4 (DPP4), forkhead box P3 (FOXP3), interleukin-4 receptor (IL4R), Jun Proto-Oncogene, AP-1 Transcription Factor Subunit (JUN), Mitogen-activated protein kinase 14 (MAPK14), MHC class I polypeptide-related sequence B (MICB), and transforming growth factor beta 1 (TGFB1). All genes had an increased methylation pattern in cells treated with endo PF compared to the media control. The exception was MAPK14, which decreased in methyl-To assess changes in promoter methylation patterns in PF treated cells, a global DNA methylation array of genes involved in inflammation and autoimmunity was performed. The heat map in Figure S2A presents a range (from 0 to 100) of "M", the fraction of input genomic DNA containing 2+ methylated CpG sites in the targeted region of a gene. Genes that were shown to be impacted by DNA methylation by having significant *p*-values (<0.05) (Figure S2B) were C-C Motif Chemokine Ligand 25 (CCL25), Cluster of Differentiation 8a (CD8A), CCAAT Enhancer Binding Protein Beta (CEBPB), Dipeptidyl peptidase 4 (DPP4), forkhead box P3 (FOXP3), interleukin-4 receptor (IL4R), Jun Proto-Oncogene, AP-1 Transcription Factor Subunit (JUN), Mitogen-activated protein kinase 14 (MAPK14), MHC class I polypeptide-related sequence B (MICB), and transforming growth factor beta 1 (TGFB1). All genes had an increased methylation pattern in cells treated with endo PF compared to the media control. The exception was MAPK14, which decreased in methylation in the endo PF treated cells. FOXP3 M values were 54.02% in endo PF treated cells (*p* < 0.0001), 26.54% in control PF treated cells (*p* = 0.0151), and 0.23% in media

ation in the endo PF treated cells. FOXP3 M values were 54.02% in endo PF treated cells

Bisulfite sequencing will be used in the future to better understand the methylation pat-

Our laboratory has been studying mechanisms leading to endometriosis and pain experienced by endometriosis patients [55–58]. This study stemmed from our previous investigations into the miRNA profile of endometriosis tissues and PF treated cells [33]. Nineteen percent of differentially expressed miRNAs in endo tissues targeted JARID2. Despite the global downregulation seen in the micronome of endometriotic tissues [33],

terns of sample DNA.

**3. Discussion** 

control. Bisulfite sequencing will be used in the future to better understand the methylation patterns of sample DNA.
