However, treatment with a combination of progesterone and estrogen did not modulate expression levels of these proteins (see Figure 4C,D for Caco2 cells, Supplementary Figure S5A,B for HCT116 cells). reduce pro-inflammatory cytokine production in intestinal epithelial models. Conclusion: Our study shows that estrogen and progesterone alleviate ER stress, decrease pro-inflammatory cytokine production, stimulate wound healing, and increase barrier function of epithelial cells. Combined, these data suggest that pregnancy hormones BMS-214662 can have beneficial effects on disease activity by positively modulating the intestinal epithelial lining. (expression. primers were used as control. For each sample 10 uL SYBRTM Select Grasp Mix and 0.5 nM primer was used. All experiments were performed a minimum of 3 times. 2.4. Scratch Assay Scratch assays were performed on Caco2 and HCT116 cell lines as described previously . In short, cell monolayers were scratched with a pipette tip, washed twice, and treated with 1 M BMS-214662 estrogen and/or progesterone. Photographs were taken (Axiovert200 M microscope; Carl Zeiss BV, Sliedrecht, The Netherlands) to analyze the percentage of open wound area at 24 h (ImageJ software; US National Institutes of Health, Bethesda, MD, USA). Five impartial wells were analyzed per condition, with two measure-sites per scratch. 2.5. MTT Cell viability was assessed using MTT assays as described previously . Cells were treated with estrogen and/or progesterone (Sigma Aldrich, St Louis, MA, USA). After BMS-214662 24 h, 48 h and 72 h, cells were incubated with 5 mM MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma Aldrich, St Louis, MA, USA) for 3 h and colorimetric changes were measured using a microplate reader (Model 680XR Bio-Rad, Hercules, CA, USA) at 490 and 595 nm. A minimum of three independent experiments were performed with each measurement in performed in duplicate. 2.6. TEER Transepithelial resistance was measured using the Epithelial Voltohmmeter (EVOM2, Sarasota, FL, USA). Caco2 and T84 cells were seeded in a Transwell (6,5 mm insert, Costar, Kennebunk, ME, USA) and grown to confluency. Cells were subsequently stimulated with Icam1 10 M estrogen and/or progesterone and resistance was measured at 0, 24 and 72 h. A minimum of three impartial measurements were performed for time point. 2.7. Enzyme Linked Immunosorbent Assay (ELISA) Caco2 cells were plated at 900,000 per well in 24-well plates. Upon attachment to the plate, cells were treated as described in the text and supernatant was harvested after 24 h. Experiments with cells were performed four times and experiments with organoids were performed nine times. Cytokine levels in supernatants from intestinal cells and organoids were determined by ELISA (Ready-SET-Go!? eBioscience, San Diego, CA, USA) as per manufacturers instructions. All samples were tested in duplicate in the ELISAs. 2.8. Western Blotting Caco2, HCT116 cells and organoids were treated with tunicamycin (0.5M) in the presence or absence of 10 M estrogen and/or progesterone. Cells were lysed in Laemmli buffer (100 mM TrisCHCl (pH 6.8), 200 mM dithiothreitol, 4% SDS, 0.1% bromophenol blue, 20% glycerol, and 2% DTT) and proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck chemicals BV, Amsterdam, the Netherlands) as described . Membranes were blocked in 50% odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) in PBS/0.05% Tween-20 and incubated overnight at 4 C with primary antibody. After washing in PBS-Tween, membranes were incubated with IRDye? antibodies (LI-COR Biosciences, Lincoln, NE, USA) for 1 h. Detection was performed using an Odyssey reader and analyzed using manufacturers software. All experiments were performed a minimum of three times. 2.9. Statistical Analysis For in vitro and ex vivo experiments, normality of distribution was assessed with DAgostino BMS-214662 and Pearson Omnibus normality test. When passing normality test or when there were insufficient numbers to calculate normality, parametric.