Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: β-Arrestin2 is a critical component of the GPCR–eNOS signalosome

doi: 10.1073/pnas.1922608117

Figure Lengend Snippet: Superresolution imaging of β-Arr2/eNOS in normal and injured SECs. SECs were isolated form normal (A) and injured (BDL, B) rat livers. β-Arr2 and eNOS were immmunolabeled with mouse monoclonal anti–β-Arr2 and rabbit polyclonal anti-eNOS antibody. β-Arr2 (Alexa Fluor 647, green) and eNOS (Alexa Fluor 568, magenta) were visualized in horizontal cross-section (X-Y), and in cross-section (X-Z) with an SR-200 inverted microscope (Vutara, Inc.) (60×/1.2 NA Olympus water immersion objective). Samples were imaged based on the SML biplane FPALM technology as described. Representative white field (Left) with the portion of the cell that is imaged at high resolution; X-Y section with eNOS labeled in magenta and β-Arr2 in green; and X-Z section images with β-Arr2 and eNOS colocalization appearing white from normal SECs (Upper) and injured SECs (Lower) are shown. (Scale bars, 1 μm.)

Article Snippet: Samples were imaged based on the single molecule localization (SML) biplane fluorescence photoactivation localization microscopy (FPALM) technology as described ( 27 , 28 ) and data were analyzed using Vutara SRX software (version 3.16).

Techniques: Imaging, Isolation, Inverted Microscopy, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: β-Arrestin2 is a critical component of the GPCR–eNOS signalosome

doi: 10.1073/pnas.1922608117

Figure Lengend Snippet: ET-1 activation of β-Arr2 and eNOS. (A) SECs from normal (Left two panels) and BDL injured (Right two panels) rat livers were cultured for 24 h and exposed to ET-1 (20 nM) for 30 min. Cells were fixed and labeled with antibody to β-Arr2 (Upper red) and nuclei were labeled with DAPI (Lower, blue). Representative images (of more than 10 others) of single and merged channels are shown. (Scale bars, 10 μm.) (B) SECs isolated from normal rat livers (Left two panels) and BDL injured (Right two panels) rat livers were cultured for 24 h and exposed to ET-1 as in A. β-Arr2 and eNOS were immmunolabeled with anti–β-Arr2 and anti-eNOS antibodies. β-Arr2 (Alexa Fluor 647, green) and eNOS (Alexa Fluor 568, magenta) were visualized in horizontal cross-section (X-Y), in cross-section (X-Z) with an SR-200 inverted microscope (Vutara, Inc.) (60×/1.2 NA Olympus water immersion objective). Samples were imaged based on the SML biplane FPALM technology as described. Representative X-Y section (Upper) and X-Z (Bottom) section images from control (Left) and exposed to ET-1 (Right) are shown. (Scale bar, 1 μm.) (C) β-Arr2 and eNOS colocalization in normal and injured SECs as in B was quantified as in Materials and Methods; the changes in normal and injured SECs are presented graphically (1 for complete and 0 for no colocalization [n = 3/group]). (D) SECs isolated from β-Arr2 WT and KO mice were exposed to ET-1 (20 nM) for 30 min. Cell lysates were subjected to immunoblotting with the indicated antibodies. Specific bands corresponding to P-eNOS were quantified and are presented as the ratio of P-eNOS to eNOS, shown on the Right (n = 3/group). (E) eNOS enzymatic activity was measured in cell lysates treated as in D and NOS activity was normalized to that of control cells from β-Arr2 WT mice without ET-1 exposure and is presented graphically (the activity in SECs from β-Arr2 WT mice without ET-1 exposure was set at 100; n = 3/group). Statistical significance for C–E was evaluated by ordinary one-way ANOVA. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.005 for differences between indicated groups; n.s., no significant difference.

Article Snippet: Samples were imaged based on the single molecule localization (SML) biplane fluorescence photoactivation localization microscopy (FPALM) technology as described ( 27 , 28 ) and data were analyzed using Vutara SRX software (version 3.16).

Techniques: Activation Assay, Cell Culture, Labeling, Isolation, Inverted Microscopy, Control, Western Blot, Activity Assay

Journal: Oncology Letters

Article Title: The expression of trefoil factor family member 2 in increased at an acidic pH

doi: 10.3892/ol.2024.14345

Figure Lengend Snippet: Effect of acidic medium on TFF expression. The graph provides a visual representation of the relative quantification, standardized by the expression levels of each gene, with pH 7.4 expression serving as a control. Subsequently, P-values were calculated using relative quantification values through one-way analysis of variance followed by Dunnett's multiple comparisons test. (A) PDK4 , expressed under acidic conditions, was used as an expression marker. PDK4 was significantly upregulated in cells cultured at pH 6.5 and 6.8 compared to pH 7.4 as a control (**P<0.01). (B) TFF1 and TFF2 expressions were significantly increased at both pH 6.5 and 6.8 compared to pH 7.4. TFF2 expression exhibited the greatest increase (**P<0.01 and ***P<0.0001). (C) The graphs were standardized using the TFF2 expression in cells cultured for 1 h at pH 7.4 as a control. TFF2 expression increased time-dependently, with significant augmentation observed in the acidic media after 24 h compared to the control (***P<0.0001). Each assay was performed in quadruplicate. Data are presented as the mean ± standard error. TFF , trefoil factor family member; GAPDH , glyceraldehyde-3-phosphate dehydrogenase; PDK4 , pyruvate dehydrogenase kinase isozyme 4.

Article Snippet: After 10 min of blocking, the cells were incubated with the primary antibodies anti-TFF2 (Protein tech, Rosemont, IL, USA, 13681-1-AP, 1:100 dilution) for 1 h at 25°C room temperature.

Techniques: Expressing, Quantitative Proteomics, Control, Marker, Cell Culture

Journal: Oncology Letters

Article Title: The expression of trefoil factor family member 2 in increased at an acidic pH

doi: 10.3892/ol.2024.14345

Figure Lengend Snippet: Effect of acidic medium on TFF2 expression in other cell lines. Cells were cultured for 48 h in medium with pH of 6.5 and 6.8. TFF2 expression at pH 7.4 was used as a control for relative quantification. P-values were calculated using relative quantification values via one-way analysis of variance followed by Dunnett's multiple comparisons test. GAPDH was used as an endogenous control. (A) Comparison of PDK4 expression in Caco-2 colon cancer, HeLa cervical carcinoma and HepG2 hepatocellular carcinoma cells cultured in acidic media and in each cell cultured at pH 7.4 for 48 h as a control (***P<0.0001). (B) Comparison of TFF2 expression in Caco-2, HeLa, and HepG2 cells cultured in acidic media and in each cell cultured at pH 7.4 for 48 h as a control (**P<0.01, ***P<0.0001). Each assay was performed in quadruplicate. Data are presented as the mean ± standard error. TFF2 , trefoil factor family member 2; GAPDH , glyceraldehyde-3-phosphate dehydrogenase; PDK4 , pyruvate dehydrogenase kinase isozyme 4.

Article Snippet: After 10 min of blocking, the cells were incubated with the primary antibodies anti-TFF2 (Protein tech, Rosemont, IL, USA, 13681-1-AP, 1:100 dilution) for 1 h at 25°C room temperature.

Techniques: Expressing, Cell Culture, Control, Quantitative Proteomics, Comparison

Journal: Oncology Letters

Article Title: The expression of trefoil factor family member 2 in increased at an acidic pH

doi: 10.3892/ol.2024.14345

Figure Lengend Snippet: Immunohistochemistry staining of DLD-1 colon cancer and Caco-2 colon cancer cells. (A) DLD-1 cells and (B) Caco-2 cells. Cells were cultured under normal pH (pH 7.4) and acidic medium (pH 6.5). Images show (a) Negative control without primary antibody and with secondary antibody and (b) Immunohistochemistry staining with TFF2 primary antibody. Under ×100 magnification microscope observation (scale bar=10 µm). (c) Quantification of TFF2 specific DAB staining was performed using ImageJ software (mean ± standard error; n=4). *P<0.05 (nonparametric Mann-Whitney U test).

Article Snippet: After 10 min of blocking, the cells were incubated with the primary antibodies anti-TFF2 (Protein tech, Rosemont, IL, USA, 13681-1-AP, 1:100 dilution) for 1 h at 25°C room temperature.

Techniques: Immunohistochemistry, Staining, Cell Culture, Negative Control, Microscopy, Software, MANN-WHITNEY