2 2 amino di  (MedChemExpress)

Journal: Biomedicines

Article Title: Unraveling the Role of Metabolic Endotoxemia in Accelerating Breast Tumor Progression

doi: 10.3390/biomedicines13081868

Figure Lengend Snippet: Activation of STAT-3 and AKT signaling pathways in BC cells under ME conditions in vitro. ( A , B ) MDA-MB-231 cells were serum-starved overnight and then either remained untreated (Cont) or incubated with LPS (0.1 ng/mL, mimicking ME conditions) for 0.5 h and 2 h. At the indicated time points, cells were harvested, and cell lysates containing equivalent amounts of total protein were immunoblotted using antibody specific for phospho-STAT3 (p-STAT3), phospho-AKT (p-AKT), total STAT3, total AKT, or total Actin (the Actin bands presented in panels A and B are from the same membrane). Band intensity was quantified using ImageJ software and intensity ratio (phospho/total) was calculated. The data shown are representative of three independent experiments. Mann–Whitney ** p < 0.01; Student’s t -test * p < 0.05. ( C ) MDA-MB-231 cells were seeded on coverslips in quadruplicates and untreated (Cont) or stimulated with LPS (0.1 ng/mL, mimicking ME conditions) for 0.5 h and 2 h. The cells were then stained with anti-pSTAT3 antibody (green). Cell nuclei were counterstained with DRAQ5 (red). Overlay (orange, indicated by white arrowheads) represents cells positive for nuclear-localized p-STAT3. ( D ) Quantification of the degree of association between p-STAT3 and DRAQ5 staining within MDA-MB-231 cells was performed using colocalization tool of ImageJ software, microscopic field = 0.0076 mm 2 ; ≥100 cells per coverslip were analyzed, four coverslips/condition. Error bars, ±SE. Student’s t -test * p < 0.05.

Article Snippet: Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI) or 1,5-bis{[2-(di-methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione (DRAQ5) (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Activation Assay, Protein-Protein interactions, In Vitro, Incubation, Membrane, Software, MANN-WHITNEY, Staining

Journal: Biomedicines

Article Title: Unraveling the Role of Metabolic Endotoxemia in Accelerating Breast Tumor Progression

doi: 10.3390/biomedicines13081868

Figure Lengend Snippet: Increased levels of IL-6 expressing macrophage infiltration in BC tumor tissue are associated with metabolic endotoxemia in vivo. ( A ) MDA-MB-231 cells were injected sc. in nude mice implanted sc. with the Alzet osmotic minipump filled with either saline (control) or LPS (ME). Representative photographs of tumor tissue sections processed for colocalization immunofluorescent analysis using anti-IL-6 (orange) and anti-F4/80 (macrophage-specific marker, green) antibodies, as described in Methods. Cell nuclei were counterstained with DRAQ5 (blue). Overlay (pink, arrowheads) represents IL-6 positive-F4/80 positive macrophages. Scale bar: 20 µm. ( B ) Quantification of the degree of association between IL-6 and F4/80 staining within tumor tissue was performed using colocalization tool of Zen software (Carl Zeiss). Bar graph shows the Pearson’s correlation coefficient values for IL-6/F4/80 colocalization, * p < 0.04.

Article Snippet: Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI) or 1,5-bis{[2-(di-methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione (DRAQ5) (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, In Vivo, Injection, Saline, Control, Marker, Staining, Software