Fig 2: SREBP2 represses KISS-1 expression by binding to the KISS-1 promoter.A Half-lives of KISS-1 protein expression in CTRi and SCAPi VSMCs after treatment with 50 µg/ml cycloheximide (CHX) and harvesting at different time points as indicated. B Relative KISS-1 mRNA expression levels in CTRi and SCAPi VSMCs as measured by qRT?PCR. C Immunofluorescence staining of SREBP2 in CTRi and SCAPi VSMCs. Scale bar = 10 µm. D Immunoblot analysis and quantification of SREBP2 protein expression in the nucleus and cytoplasm of CTRi and SCAPi VSMCs. E A putative SREBP2 binding site was predicted in the promoter region of KISS-1 (422 to 431) by the JASPAR database. F Agarose electrophoresis following ChIP to determine the SREBP2 binding site in the KISS-1 promoter. G The relative luciferase activities of KISS-1 were detected after cotransfection with the pGL3-basic luciferase reporter plasmid vector and different contents of the constructed SREBP2 plasmid for 48 h. H Relative mRNA expression levels of SREBP2, KISS-1, MMP9 and VEGF in CTRi and SREBPi VSMCs as measured by qRT?PCR. I Secretion concentrations of KISS-1 in culture supernatant as measured by ELISA in VSMCs treated with CTRi and SREBP2i. J Representative images of wound-healing migration assays in VSMCs treated with CTRi and SREBP2i for 0 h and 48 h. K Schematic representation of how SCAP in VSMCs affects embryonic angiogenesis through negative regulation of KISS-1. All experiments were repeated at least 3 times. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P values were calculated by Student’s t test.

Fig 3: Ablation of SCAP in VSMCs inhibits endothelial cell function through intercellular communication.A Heatmap of the related molecules in the VEGF signaling pathway from the placentas of SCAP+/+ and SCAPfl/fl mice. B Secretion concentrations of KISS-1 in culture supernatant as measured by ELISA in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. C Concentrations of VEGF in the culture supernatant as measured by ELISA in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. D Immunoblot analysis of VEGF and ICAM protein expression in HUVECs cocultured with VSMCs of different groups. E Statistical analysis of the relative protein expression levels in (C). F Representative images of the EdU assay of HUVECs co-cultured with supernatant from VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. G Statistical analysis of the relative cell proliferation rate in (F). H Representative images of the Transwell migration assay of HUVECs co-cultured with supernatant from VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. I Statistical analysis of the relative cell numbers per field (%) in (C). J Representative images of tube formation assays of HUVECs co-cultured with supernatant from VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. K Quantitative analysis of number of nodes in (J). L Quantitative analysis of number of Junctions in (J). All experiments were repeated at least 3 times. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P values were calculated by Student’s t test.

Fig 4: Knockdown of KISS-1 expression rescues SCAP-ablated VSMCs from loss of proliferation and migration abilities in vitro.A Representative images of wound-healing migration assays in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i for 0 h and 48 h. B Statistical analysis of the relative VSMC migration rates in (A). C Representative images of the Transwell migration assay in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. D Statistical analysis of the relative cell numbers per field (%) in (C). E Representative images of the EdU assay in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. F Statistical analysis of the relative cell proliferation rate in (E). G Immunoblot analysis and quantification of SCAP, N-SREBP2, KISS-1, GPR54, P-MAPK, MAPK, P-ERK, ERK, P-JNK and JNK protein expression in VSMCs treated with CTRi, SCAPi, SCAPi and KISS-1i. All experiments were repeated at least 3 times. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P values were calculated by Student’s t test.