Fig. 2

AMF interacts with GPER-1. a, A genome-wide expression microarray was used to detect the changes in gene expression by GPCRs under AMFR silencing conditions, and the histogram of the top 10 pathways per the signaling pathway enrichment results is shown. GPCR-related pathways are individually annotated in red. b The heatmaps of the same trend changes observed in Ishikawa and SPEC-2 cells (> 1.5-fold) were plotted using Mev software (MeV_4_6_0), including 38 GPCR genes (25 upregulated and 14 downregulated). c. Analysis of the gene interactions of 25 upregulated GPCR genes. The results showed that GPER-1 expression was significantly associated with AMF, as shown by the String database. d. Cells containing the plasmids encoding PBT3-SUC-GPER-1-14 and PPR3-N-GPI-3 were able to grow in SD medium lacking histidine and adenine. Deletion of the transactivation domain as a negative control completely abolished the growth of clones, and replacement of PNubG-Fe65 and PTsu2-App was used as a positive control in the selective medium. e. Serum-starved Ishikawa and SPEC-2 cells were treated simultaneously with AMF and DTSSP for 1 h at 4 °C to cross-link extracellular proteins. Coimmunoprecipitation of lysates was conducted as indicated, and the precipitates were analyzed by Western blot analysis, which allows for the identification of interacting proteins. AMF-treated and cross-linked cells were used for coimmunoprecipitation with anti-AMF (top) or anti-GPER-1 (bottom). f. Colocalization of GPER-1 and AMF in serum-starved EC cells, which were treated with exogenous AMF (10 ng/ml) for 24 h. The merge is shown as GPER-1 (red), AMF (green) and DAPI (nuclear stain, blue). Control indicates conditioned medium alone. g and h. Cells were treated with AMF (1 ng/ml; 0, 1, or 5 min), and the expression of AMF and GPER-1 was detected using immunofluorescence. i. Western blots were used to detect the expression of GPER-1 protein in both the cell membrane and cytoplasm to further confirm the results. j. Prediction of interaction of AMF-GPER-1 docking, red and green indicate AMF and GPER-1 respectively