Fig 1: GPER agonist G-1 and venetoclax synergistically inhibit leukemic cell survival through apoptosis and pyroptosis.A–C CCK-8 assay of cell viability in the cell lines and primary blasts treated with G-1 and VEN, alone or in combination for 48 h, CI values were calculated using CompuSyn software. D–F FCM of apoptosis in the cell lines, primary blasts treated with G-1 (1 µM) and VEN (OCI-AML2; 0.02 µM, KG1a; 0.2 µM, AML#7; 0.2 µM), alone or in combination for 24 h. G Western blotting of these apoptosis related proteins. H Western blotting of Cyto C in the mitochondria fractions and cytosolic fraction of the cells treated with indicated drugs for 24 h. I Representative light microscopy images of the same treated cells as in Fig. 3D, E. The red arrowheads indicated the characteristic balloons on the cell membrane (Scale bar: 50 µm). J Representative transmission electronic micrographs of the leukemic cells (Scale bar: 2 µm). The data are expressed as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2: Pharmacologic activation of GPER inhibits leukemic cell survival via mitochondrial-related apoptosis.A CCK-8 assay of cell viability in the leukemic cells treated with corresponding concentrations of GPER agonist G-1 for 48 h, IC50 values were calculated on basis of drug concentrations that causes 50% cell viability. B Leukemic cells were treated with 1 µM G-1 for the corresponding times. C CCK-8 assay of cell viability in the primary blasts and PBMNCs from HD treated with G-1 for 48 h. D, E FCM of apoptosis in the cell lines treated with G-1 for 24 h, and western blotting of the indicated proteins. F Western blot analysis of Cyto C in the mitochondria fractions and cytosolic fraction of the cells treated with indicated drugs for 24 h. HSP60 or GAPDH were used as an internal control. G Western blot analysis of GPER in OCI-AML2 cells infected with two independent sh RNAs targeting GPER. H FCM of apoptosis in the cells treated with 1 µM G-1 for 24 h. The data are expressed as the mean ± SD (n = 3). *p < 0.05, **p < 0.01. ns, not significant.
Fig 3: NEH inhibits the GPR30-mediated PI3K/AKT signalling pathway. (a) Western blot analysis on the influence of NEH on GPR30 and molecules on the PI3K/AKT signalling pathway. The data in (b) were presented as the mean ± SEM. ***P < 0.001, compared with the control group. (c–f) Western blot analysis on the effects of NEH, G15, and G1 on the molecules of the GPR30/PI3K/AKT signalling pathway in GC cells. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group or NEH-treated group.
Fig 4: GPER expression in the prostate tissue. Immunohistochemistry (A) and western blot (B) analysis of proteins in the prostate tissue under different conditions. *p<0.05, **p<0.01, ***p<0.001.
Fig 5: Estradiol modulates ASIC1a through its receptor GPER1.(A) Western blot analysis of the protein expression of ASIC1a with or without for estradiol (500 nmol/mL), MPP (20 mmol/L), and G15 (15 µmol/L). (B) Western blot analysis of ERa and GPER1 protein expressions in chondrocytes that without transfection or transfected with negative control (NC) or siRNA for ERa or GPER1. (C) Western blot analysis of the protein expression of ASIC1a with or without for estradiol (500 nmol/mL) and ERa-siRNA or GPER1-siRNA. (D–F) Chondrocytes in serum-free confluent monolayer cultures were stimulated by with increasing concentrations of G1 (0–1000 nmol/L) and levels of ASIC1a, GPER1, Beclin1, LC3 and p62 proteins were measured by Western blot. Data are presented as mean ± SEM, *P < 0.05, versus pH + E2 group, ^P < 0.05, ^^P < 0.01 versus the NC group, $P < 0.05, $$P < 0.01, $$$P < 0.001 versus 0 nmol/L.
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