Fig. 3

AMF activates the PI3K signaling pathway to promote EC cell growth via GPER-1. a. Growth curve by RTCA assay. Cells were seeded at a low density (2000 per well) and grown with exogenous AMF stimulation for 5 days. Fresh medium with AMF (10 ng/ml) was provided every day (points, mean of triplicate determinations; bars, SD). b. shGPER-1 and control cells were seeded in 3D culture for spheroid formation and were photographed at day 14 in culture (representative images are shown; 400× for the inserts, 200× for all others). c. Quantification of the number (left) and relative size (right) of the spheroids. d. For the identification of the target proteins upregulated by GPER-1 overexpression with the simultaneous silencing of AMFR, proteomics analysis using an iTRAQ reagent and QSTAR Elite Hybrid LC-MS/MS was performed. The labeled digests were analyzed using Nano LC-MS/MS. The distribution of enriched KEGG pathways associated with upregulated proteins is shown. e. The upregulated expressed proteins were enriched for biological process. f. The pathway interaction network was based on KEGG pathway enrichments and is shown in the middle as interactions with high confidence scores. g. The effect of wortmannin on AMF-GPER-1-induced cell proliferation. Cells treated as described above were then counted using RTCA assays. Data represent the mean ± SD of the three independent experiments. h. EC cells with overexpression of GPER-1 or shGPER-1 and AMFR silencing were serum-starved for 16 h and stimulated with purified AMF (10 ng/ml); p-ERK and p-AKT levels were monitored by Western blot after 15 min, and β-actin was used as a loading control. i and j. Cell apoptosis and cell cycle profiles were analyzed by fluorescence-activated cell sorting (FACS). (*P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant, one-way ANOVA)