e2 glow  (Jena Bioscience)

Journal: Scientific Reports

Article Title: Estradiol/GPER affects the integrity of mammary duct-like structures in vitro

doi: 10.1038/s41598-020-57819-9

Figure Lengend Snippet: Effect of E2 on a 3D model of the milk duct using MCF-10A cells. ( a ) Representative confocal images of MCF-10A cells in a 3D culture through the middle acini, which were treated with E2 (32 nM, left two panels) or control (0 nM, right panel) for 7 days. The basement membrane was examined via immunofluorescence staining using laminin V antibody (red); cell junctions were evaluated using pan-cadherin antibody (green). The reconstructed images of the acini structures by confocal microscopy are shown at the bottom with Hoechst (blue) and laminin V (red) staining. Arrows indicate the collapsed portion of the basement membrane. Scale bars = 5 μm. ( b ) The basement membrane was stained using anti-laminin V antibody, and the percentage of acini with disrupted basement membranes was calculated. Three independent experiments (32 nM E2; 54.5% (n = 55), 50% (n = 48), 43.8% (n = 57), 0 nM E2; 23.1% (n = 52), 22.2% (n = 54), 10% (n = 50)) were performed. Bars represent +/−SD. DATA were analyzed using a Mann-Whitney U test. *p values less than 0.05 were considered statistically significant. ( c ) Representative SEM images of MCF-10A cells in a 3D culture treated with 32 nM E2 for 72 h. SEM images are shown in Matrigel matrix (blue) and basement membrane (pink). ( d ) Western blotting of GPER-expressing cell lysates (MCF-7, U2OS, MCF-10A, T47D, and MDA-MB-231) (left). MCF-7 and MCF-10A cell lysates were further probed for ERα expression. ( e) Immunohistochemical analysis of GPER expression (green) and the basement membrane (laminin V, red) in normal human breast, ductal carcinoma in situ , and invasive tissue. Blue, Hoechst staining. Scale bars = 10 μm. ( f ) Immunofluorescence analysis of MCF-10A cells following treatment with fluorescently labeled E2 (green) for 5 min to examine the colocalization of E2 and GPER (red). Blue, Hoechst staining. Scale bars = 20 μm. ( g ) Binding of E2-Glow to FLAG-GPER which was expressed in 293 T cells and immunoprecipitated with FLAG antibody. 1.27 ± 0.68 μM E2-Glow was bound to FLAG-GPER. Five independent experiments were performed. Bars represent +/−SD. The presented blots were cropped. Full-length blots are presented in Supplementary Fig. .

Article Snippet: E2-Glow, fluorescently labeled E2, was purchased from Jena Bioscience .

Techniques: Control, Membrane, Immunofluorescence, Staining, Confocal Microscopy, MANN-WHITNEY, Western Blot, Expressing, Immunohistochemical staining, In Situ, Labeling, Binding Assay, Immunoprecipitation

Journal: Scientific Reports

Article Title: Estradiol/GPER affects the integrity of mammary duct-like structures in vitro

doi: 10.1038/s41598-020-57819-9

Figure Lengend Snippet: E2-induced IL-1β secretion and pyroptosis. ( a ) IL-1β ELISA of MCF-10A cells examined for the concentrations of secreted IL-1β in the cell culture media following treating the cells with 0–128 nM E2 for 60 h. Four independent experiments were performed. Bars represent +/−SD. ( b ) IL-1β ELISA of MCF-10A cells showing the concentrations of secreted IL-1β in the supernatant following treatment with 32 nM E2 for 1–72 h. Three independent experiments were performed. Bars represent +/−SD. ( c ) Representative immunofluorescence images of MCF-10A cells following treatment with fluorescently labeled E2 (green) and MitoTracker Red CMXRos (red) to examine the localization of E2 in mitochondria. E2 localized to mitochondria with or without GPER. Blue staining, Hoechst. Scale bars = 5 μm. ( d ) Cell-based ROS assay to measure ROS in MCF-10A cells following treatment with 32 nM E2 for 15 or 30 min. Antimycin A, an inhibitor of complex 3 of the mitochondrial electron transport chain, was included as a positive control for ROS production, and N-acetyl cysteine was included as an antioxidant control. Four independent experiments were performed. Bars represent +/−SD. DATA were analyzed using a Mann-Whitney U test. *p values less than 0.05 were considered statistically significant. ( e ) Caspase-1 inflammasome assay was used to measure caspase-1 activity in MCF-10A cells after adding 32 nM E2 for 24 h. YVAD-CHO was used as a caspase-1 inhibitor. Three independent experiments were performed. Bars represent +/−SD. DATA were analyzed using a Mann-Whitney U test. *p values less than 0.05 were considered statistically significant. ( f ) Western blotting of MCF-10A cells transfected with p3xFLAG-GSDMD to investigate full-length FLAG-GSDMD and cleaved FLAG-GSDMD (31 kDa) using the FLAG antibody following treatment with 32 nM E2 for 0–4 h. ( g ) Western blotting of MCF-10A cells showing endogenous WT-GSDMD (full length) and cleaved GSDMD following treatment with 32 nM E2 for 0–4 h using an antibody that recognizes the GSDMD-N-terminal. ( h ) Confocal images of MCF-10A cells treated with 32 nM E2 for 48 h (bottom) or without 32 nM E2 (up) and stained with the GSDMD antibody (green) to investigate GSDMD (N-terminal) distribution on the plasma membrane. Two confocal cellular cross sections are shown. Scale bars = 10 μm. ( i ) SEM electron microscopy imaging of MCF-10A cells treated with 32 nM E2 for 72 h showing pyroptotic bodies on the surface of the plasma membrane. Scale bars = 1 μm. The presented blots were cropped. Full-length blots are presented in Supplementary Fig. .

Article Snippet: E2-Glow, fluorescently labeled E2, was purchased from Jena Bioscience .

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Immunofluorescence, Labeling, Staining, ROS Assay, Positive Control, Control, MANN-WHITNEY, Activity Assay, Western Blot, Transfection, Clinical Proteomics, Membrane, Electron Microscopy, Imaging