Rgfp0966

Cell viability was determined by performing the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay (Promega) according to the manufacturer’s instructions. Briefly, iHEECs/Luc or primary human endometriotic stromal cells were counted and plated in 96-well plates, and relative viability rates were evaluated at the indicated time points after treatment with n-butyrate, 100 nM GLPG0974, 100 nM MB, 20 μM SAHA, 500 nM TSA, 3 μM MS-275, 3 mM Valproic acid, or 10 μM RGFP0966 (all from Sigma-Aldrich). For the GPRs study, the cells were pre-treated for 1 h with 100 nM GLPG0974, 100 nM MB, or both, then treated with 2 mM n-butyrate for 24 h. For knockdown experiments, after 48 h of siRNA transfection, iHEECs/Luc were re-plated in 96-well plates at 5 × 10³ cells per well. After 24 h, cells were treated with 2 mM sodium butyrate (Sigma-Aldrich) for 0, 24, 48, or 72 h. At each time point, cell viability was determined by the MTT assay. In all cases, 15 μl of MTS (dye solution) reagent (Promega) was added to each well and incubated for another 2 h. After addition of 100 μl of Solubilization Solution, absorbance was measured at 570 nm with 650 nm as a reference wavelength in a 96‐well plate reader. The experiments were performed three times each with three to five technical replicates.

From day 0 to day 21 after endometriosis induction, mice were provided drinking water containing 300 mM sodium acetate (36 (link)), sodium propionate (36 (link)), or sodium butyrate (19 (link)) (all from Sigma-Aldrich) (36 (link)). These solutions were changed every week. The GPR43 antagonist GLPG0974 (10 mg/kg) and GPR109A inhibitor MB (10 mg/kg) were dissolved in dimethyl sulfoxide (vehicle) with 30% PEG-300 (Sigma-Aldrich) and administered via daily intra-peritoneal injection. Trichostatin-A (1 mg/kg), SAHA (25 mg/kg), MS-275 (20 mg/kg), valproic acid (500 mg/kg), and RGFP0966 (25 mg/kg) (all from Sigma-Aldrich) were dissolved in dimethyl sulfoxide with 30% PEG-300 and administered from day 0 to day 21 via daily intra-peritoneal injection. Similar amount of dimethyl sulfoxide with 30% PEG-300 was administered as vehicle.