Fig 1: NrCAM from progestin-pretreated ESCs mediated the inhibitory effect of ESCs on EC cell proliferation.A Cluster analysis of MPA-regulated genes encoding secretory proteins in ESCs. ESCs were treated with 10 μM MPA or ethanol (EtOH) for 6 h before RNA sequencing. B mRNA levels of NrCAM, BMP2, WISP1, and ITGA10 were significantly upregulated in ESCs after MPA treatment. Silencing PR expression with siPGR in ESCs weakened MPA-induced upregulation of these four proteins. ESCs or ESCs-siPGR were treated with or without 10 μM MPA for 6 h. Eleven candidate gene mRNA levels were reevaluated by real-time PCR. C Exogenous NrCAM inhibited EC cell proliferation in a dose-dependent manner. Ishikawa and ECC-1 cells were treated with 0, 1, 10, 100, and 1000 ng/mL NrCAM for 48 h before CCK-8 assays. D Exogenous NrCAM inhibited EC cell proliferation in a time-dependent manner. Ishikawa and ECC-1 cells were treated with 1000 ng/mL NrCAM for 24, 48, and 72 h before CCK-8 assays. E MPA promoted NrCAM protein expression in ESCs in a dose-dependent manner. NrCAM expression was detected by western blotting. ESCs were treated with 0, 5, 10, and 20 μM MPA for 48 h. F MPA promoted NrCAM secretion in ESCs by ELISA. ESCs were treated with MPA at the indicated dose for 48 h (left) or 10 μM MPA for 24, 48, or 72 h (right). The CM extracted from ESCs was collected to measure NrCAM concentration by ELISA. G MPA-induced NrCAM protein expression was attenuated by silencing PGR in ESCs. ESCs or ESCs-siPGR were treated with 10 μM MPA for 48 h before western blotting analysis. H NrCAM and MPA cotreatment had a stronger inhibitory effect on EC cell proliferation than MPA or NrCAM alone. Ishikawa and ECC-1 cells were treated with 1000 ng/mL NrCAM and/or 10 μM MPA for 48 h before CCK-8 assays. I Transfection efficiency of siRNAs targeting NrCAM was confirmed by real-time PCR and western blotting. J The inhibitory effect of ESCs on EC cell proliferation was blocked by silencing NrCAM expression in ESCs. After transfection with siNrCAM or siCtrl for 8 h, ESCs were treated with or without 10 μM MPA for 48 h, then for CM collection. Ishikawa and ECC-1 cells were treated with 10 μM MPA, CM (ESCs-siNrCAM-2) and CM (ESCs-siNrCAM-2 + MPA) for 48 h before CCK-8 assays. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant.