Fig. 8
hEMO display functional PAR2 expression. A In situ hybridization images of hEMO in 2D and 3D conformation. Signals were detected for PAR2 (F2rl1) and the epithelial marker E-cadherin (Cdh1). Scale bar: 50 µm. B mRNA expression of PAR2, ORAI1 and STIM1 in hEMO. Expression is relatively quantified compared to geometric mean of the housekeeping genes HPRT1 and PGK1. Data is shown as mean ± SEM. C-G Ca2+ microfluorimetry. In C and D representative traces of 2D hEMO stimulation with trypsin (1 µg/ml) or 2-furoyl-LIGRLO-NH2 (250 nM) are shown. In E trypsin responses were challenged with the specific PAR2 inhibitor I-191 (100 nM). F 2D hEMO treated with vehicle, without shear stress. Ionomycin (2 µM) was added at the end of each experiment as positive control. G Percentage of oscillating cells in response to trypsin (2 µg/ml), 2-furoyl-LIGRLO-NH2 (5 µM), simultaneous application of trypsin with the specific PAR2 inhibitor I-191 (100 nM) and vehicle. The presence ( +) or absence (-) of shear stress (SS) is shown. NR: no responding cells. Data is shown as mean ± SEM. N = at least 3 independent experiments on hEMO obtained from minimum two different patients, with a total minimum of at least 130 cells. H mRNA expression of decidualization markers PAEP, SPP1, CXCL14, LIF and GPX3 in hEMO. Expression is relatively quantified compared to the housekeeping gene GAPDH. Data is shown as mean ± SEM. N = 3 independent experiments. Two-way ANOVA with Dunnett’s multiple comparison test. * p < 0.05, ** p < 0.01 compared to EPC condition. Tryp = trypsin, 2-fu = 2-furoyl-LIGRLO-NH2, Iono = ionomycin. SS = shear stress