Fig 2: GPER expression pattern in different bronchopulmonary and gastroenteropancreatic neuroendocrine tumour entities. Immunohistochemical staining (red-brown colour) and counterstaining with haematoxylin. Scale bar: 100 µm (A–F). Asterisk in (D), liver metastasis (MTS) of an ileum neuroendocrine neoplasm (NEN); arrows in (D), surrounding GPER-positive non-neoplastic liver tissue.

Fig 3: Double-labelling immunohistochemical analysis of human pancreatic islets. Sections were dewaxed and microwaved in citric acid. Adjacent sections of each tissue sample were incubated with rabbit monoclonal anti-GPER antibody 20H15L21 together with mouse monoclonal antibodies against glucagon or insulin, or a rat antibody against somatostatin-14/28. Labelling for GPER was visualised using a Cy3-conjugated anti-rabbit antibody (red), and labelling for insulin, glucagon, or somatostatin-14/28 was visualised using an Alexa Fluor 488-conjugated anti-mouse or anti-rat antibody (green). Scale bar: 100 µm.

Fig 4: The GluR2-AMPAR/GPER1 ratio and molecular distance between GPER1 and GluR2-AMPAR in the membrane. (A) STORM images depicting immunolabeled AMPAR (magenta) and GPER1 (cyan) molecules on dPC12. Dashed lines delineate the borders of the neurites and somas. Scale bar = 2 µm; inset Scale bar = 0.5 µm. (B) The ratio between the number of GPER1 and AMPAR molecules (GPER1/GluR2-AMPAR) on the neurites and somas (n = 11 somas or neurites). (C1) Photomicrographs depict GPER1 immunoreactivity (visualized with STED microscopy) in dPC12 after 10 min of vehicle (left) or of 100 nM of E2 treatment (right). Scale bar = 2 µm. (C2) One 2 µm2 (between parallel white bars) and one 10 µm2 (to the left) areas were selected within each ROI for the membrane and cytoplasmic regions of each cell, respectively. Integrated density was calculated and normalized to the area. Scale bar = 0.5 µm. (D) Dual labeling of plasma membrane and GPER1 molecules defines the membrane regions (approximately 1 µm wide). Scale bar = 0.5 µm. (E) Line graph of the fluorescent intensity calculated from the magnified STED inserts (C2). (F) Integrated density graphs of GPER1 show the effect of vehicle and 100 nM of E2 treatment in the membrane and in the cytoplasm (n = 15 cells were evaluated in each group). *p < 0.05.

Fig 5: Oxygen-dependent regulation of wound healing and migration by estradiol in DLD-1 cells(A) Wound healing assay in DLD-1 cells under normoxic (20.9% O2) and hypoxic (2% O2) conditions. Representative images are shown of the scratches at time 0 and following 24h. Dashed lines indicate wound margins. Graph shows the % wound closure under each condition. Cells were treated with 10nM estradiol (E2), 1µM G1 (a GPER-selective agonist) or 5µM G15 (a GPER-selective antagonist). Mean ± SEM, n=6, ***P<0.001, ****P<0.0001. (B) Boyden chamber migration assay in DLD-1 cells under normoxic and hypoxic conditions. Representative images are shown. Cells were treated with E2, G1, G15, G15+E2 or G15+G1. Mean ± SEM, n=6, *P<0.01 E2 and G1 treatment compared to Ctl (Veh only), †P<0.01 G15 compared to G1 and E2 treatment, ‡P<0.01 G15+E2 compared to G15 treatment, §P<0.01 G15+E2 compared to E2 and G1 treatment, #P<0.01 G15+G1 compared to G15, ***P<0.0001 G15 compared to E2 and G1, †††P<0.001 G15+E2 treatment compared to E2 or G1 treatments, ‡‡‡P<0.0001 G15+G1 compared to E2 or G1 treatments.