Fig 2: NRP1 mediated the angiogenic function of Tagln2. (A) Migration and in vitro tube-forming activity of control Ealy926 cells (pLVX-ctrl) and Ealy926 cells with stable expression of TAGLN2 (pLVX-TAGLN2) subjected to a series of treatments, including knockdown of endogenous NRP1, knockdown of endogenous Tagln2, knockdown of endogenous NRP1 with exogenous replenishment of Tagln2 or knockdown of endogenous Tagln2 with exogenous replenishment of NRP1. (B, C) After modulation of TAGLN2 and NRP1 expression in ECs, levels of VEGFR2, VWF (markers of endothelial cells), NrCAM (mediating capillary outgrowth), E-cadherin, P-selectin (related to ECs adhesion and migration), Caspase 3/cleaved Caspase 3 and PARP1/cleaved PARP1 (markers of apoptosis), as well as downstream targets, including phosphorylated ERK1/2, JNK, and C-Jun, were measured by western blotting. GAPDH was used as the loading control. **P < 0.01, ***P < 0.001. ns, not significant.

Fig 3: NrCAM increased PR expression in EC cells via TET1-mediated hydroxymethylation of the PRB promoter.A Exogenous NrCAM promoted PR protein expression in EC cells. Ishikawa and ECC-1 cells were treated with 1000 ng/mL NrCAM for 48 h. B Exogenous NrCAM increased transcription of PGR and its downstream target genes HAND2 and HR2F2 in EC cells. After treatment with 1000 ng/mL NrCAM for 12 h, Ishikawa and ECC-1 cells were analyzed by real-time PCR. C NrCAM increased genomic DNA hydroxymethylation in EC cells. Ishikawa and ECC-1 cells were treated with 1000 ng/mL NrCAM for 24 h, and then 5-hmC levels of total DNAs were assessed by dot blot. D NrCAM promoted hydroxymethylation levels of the PRB promoter in EC cells. After treatment with 1000 ng/mL NrCAM for 24 h, 5-hmC levels of the PGR promoter fragment from Ishikawa and ECC-1 cells were detected by hMeDIP. E NrCAM increased TET1 and PGR mRNA levels in EC cells. Silencing TET1 reduced NrCAM-induced PR expression in EC cells. After transfecting siTET1, Ishikawa cells were treated with 1000 ng/mL NrCAM for 24 h, then were harvested for real-time PCR analysis. F Silencing TET1 blocked NrCAM-induced hydroxymethylation levels of the PRB promoter in EC cells. After transfecting with siRNAs targeting TET1, Ishikawa cells were treated with 1000 ng/mL NrCAM for 24 h. Then 5-hmC levels of the PGR promoter fragment were detected by hMeDIP. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant.

Fig 4: 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.