*4.6. Cell Transfection with miR-155 Mimic/Inhibitor*

Cells were transfected using SiPORT™ NeoFX ™ transfection agent (Cat No: AM4510, Ambion, Austin, TX, USA) as recommended by the manufacturer. In short, the SiPORT™ NeoFX ™ was diluted in Opti-MEM® Reduced Serum Media (Cat No: 31985062, Invitrogen, Carlsbad, CA, USA) and incubated for 10 min at room temperature. miR-155 mimic (Pre-miR™), inhibitor (Anti-miR™), positive control (anti-let-7c) (Cat no: 4392431, Thermo Scientific, Waltham, MA, USA), and negative control (Negative control #1) (Cat No: AM17010, Thermo Scientific, Waltham, MA, USA) were diluted in cell media (DMEM/F12, 10% FBS, 1% Pen/Strep, 1% L-glutamine) to a final concentration of 30 nM and then combined with the transfection agent and incubated for 10 min at room temperature. Transfection mixtures were added to 6-well plates and overlaid with cell suspensions. Cells were then incubated for 24 h prior to treatment with peritoneal fluid from control and endometriosis patients, as previously described. Transfection efficiency was tested by collecting cells in Qiazol and assessing miRNA expression using the miR-155 primer assay. RNA was isolated using the miRNeasy Mini Kit following the manufacturer's recommendations (Cat No: 217004, Qiagen, Gaithersburg, MD, USA). RT-qPCR was used (as previously described) to determine the expression of key downstream targets such as, JARID2 and FOXP3.

Western blots were performed in the traditional manner. Total protein was measured using a modified Lowry method. Protein (35 µg) was separated on a 4–20% Tris-HCl gradient gel (Cat No: 4561096, Biorad, Hercules, CA, USA) and transferred onto nitrocellulose membranes. After washing with Tris-buffered saline with Tween 20 (TBST), the membranes were blocked in 5% bovine serum albumin or 5% milk in TBST for 1 h, then incubated at 4 ◦C overnight with anti-rabbit antibody against JARID2 (Cat No: 13594S), FOXP3 (Cat No: 12632S), EZH2 (Cat No; 5426S), and H3K27me3 (Cat No: 9733S) (1:1000, Cell Signaling, Danvers, MA, USA) and anti-mouse against β-actin (1:4000, Cat No: A5316, Sigma-Aldrich, St. Louis, MO, USA). Anti-rabbit antibody against H4/H3 was diluted 1:20,000 (Cat No: 07-108, Sigma-Aldrich, St. Louis, MO, USA). Dilutions for primary antibodies varies from that used for WES due to the different methods used. The membranes were washed and incubated with HRP-linked anti-rabbit or anti-mouse secondary antibody (1:6000, Cat No: A6154, A4416, Sigma-Aldrich, St. Louis, MO, USA) for one hour at room temperature. After washing, membranes were developed in HRP Substrate (Cat No: WBKLS05000, Millipore, Temecula, CA, USA) and imaged using the ChemiDoc system (Cat No: 1708265, Biorad, Hercules, CA, USA). Densitometric levels of protein bands were quantified and normalized to β-actin or H3. Results were expressed as a ratio in which media alone was 1.
