Journal: Cell reports

Article Title: ΔN-Tp63 Mediates Wnt/β-Catenin-Induced Inhibition of Differentiation in Basal Stem Cells of Mucociliary Epithelia

doi: 10.1016/j.celrep.2019.08.063

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: mouse anti-Muc5B (1:500, HS) , Santa Cruz Biotech , sc-393952.

Techniques: Recombinant, RNA Library Preparation, cDNA Synthesis, SYBR Green Assay, Quantitative RT-PCR, Software

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Generation of human MUC5B [rs35705950] transgenic mice. Preparation of a construct of human MUC5B [rs35705950] recombinant bacterial artificial chromosome (BAC) using the Red/ET recombination strategy ( A ). Sequence analysis of the junction DNA of the recombinant BAC clone ( B ). The expression construct linearized with PI-SceI (Proteinase Intein-Scel Endonuclease I) was separated by pulsed-field gel electrophoresis ( C ). The gel containing the expression construct within the agarose gel was cut out without UV irradiation (upper figure of panel C ). The DNA fragments purified by electrophoretic elution and dialysis were applied to pulsed-field gel electrophoresis to confirm that the long-chain DNA fragments were purified without fragmentation (middle figure of panel C ). The DNA concentration was determined using a NanoDrop spectrophotometer (Shimazu biotech, Kyoto, Japan) lower figure of panel C ). The linearized recombinant BAC clone was microinjected into fertilized eggs of the C57BL/6J strain and the fertilized eggs were transplanted into the oviducts of pseudo-pregnant mice to get the founders ( D – F ). The marker used was M: NEB Low Range PFG marker. Red/ET, Red recombinase system (RedαRedβ proteins)/Electroporation-Transformation-cloning. Arrows indicate the bands.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Transgenic Assay, Construct, Recombinant, Sequencing, Expressing, Pulsed-Field Gel, Electrophoresis, Agarose Gel Electrophoresis, Irradiation, Purification, Concentration Assay, Spectrophotometry, Marker, Electroporation, Transformation Assay, Cloning

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Expression of the human MUC5B [rs35705950] transgene. Southern blot screening for the transgene in the offspring, identifying three transgenic mouse founders (red arrows in ( A )). Analysis of relative gene expression of the human MUC5B [rs35705950] transgene in various tissues and organs by PCR ( B ).

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Expressing, Southern Blot, Transgenic Assay, Gene Expression

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Sequential body weight changes and significant expression of the human MUC5B transgene rs35705950 in the proximal airways in the bleomycin-induced lung fibrosis model. Lung fibrosis was induced in wild-type (WT/BLM) and human MUC5B rs35705950 transgenic (h-rs35705950-Tg/BLM) mice through continuous subcutaneous administration of BLM. Control groups, consisting of WT (WT/SAL) and human MUC5B rs35705950 transgenic (h-rs35705950-Tg/SAL) mice, similarly received sterile physiological saline. Sequential body weight changes in the four groups of mice ( A ). Relative mRNA expression of the transgene in the experimental mouse groups ( B ). Immunostaining for MUC5B protein was performed using an anti-MUC5B polyclonal antibody, which cross-reacts with both human and mouse MUC5B ( C , D ). Additionally, MUC5B protein staining was conducted using an anti-human MUC5B monoclonal antibody ( E , F ). Scale bars indicate 100 µm. Data are expressed as the mean ± SD. Statistical analysis was performed using ANOVA with the Neuman-Keuls test. * p < 0.05; *** p < 0.001; **** p < 0.0001. WT, wild-type; TG, transgenic; BLM, bleomycin; SAL, saline.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Expressing, Transgenic Assay, Control, Sterility, Saline, Immunostaining, Staining

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Reduced infiltration of inflammatory cells in human MUC5B rs35705950 transgenic mice with lung fibrosis. ( A , B ) Lung fibrosis was induced in wild-type (WT/BLM) and human MUC5B rs35705950 transgenic (h-rs35705950-Tg/BLM) mice through continuous subcutaneous administration of BLM. Control groups, consisting of WT (WT/SAL) and human MUC5B rs35705950 transgenic (h-rs35705950-Tg/SAL) mice, similarly received sterile physiological saline. On the 22nd day following BLM administration, bronchoalveolar lavage fluid was collected under profound anesthesia. The total cell count and differential cell count were then assessed. Scale bars indicate 200 μm. Data are expressed as the mean ± SD. Statistical analysis was performed by ANOVA with Neuman-Keuls test. * p < 0.05, *** p < 0.001, **** p < 0.0001. WT, wild-type; TG, transgenic; BLM, bleomycin; SAL, saline.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Transgenic Assay, Control, Sterility, Saline, Cell Counting

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Reduced expression of inflammatory cytokines in human MUC5B rs35705950 transgenic mice with lung fibrosis. Inflammatory cytokines were measured in bronchoalveolar lavage fluid ( A ) and lung tissue homogenate ( B ) by immunoassays using commercially available kits and following the protocols of the manufacturers. Data are expressed as the mean ± SD. Statistical analysis was performed by ANOVA with Neuman-Keuls test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. WT, wild-type; TG, transgenic; BLM, bleomycin; SAL, saline; ns, not significant; OPN, osteopontin.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Expressing, Transgenic Assay, Saline

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Decreased expression of growth factors in human MUC5B rs35705950 transgenic mice with lung fibrosis. Growth factors were measured in bronchoalveolar lavage fluid ( A ) and lung tissue homogenate ( B ) by immunoassays using commercially available kits and following the protocols of the manufacturers. Data are expressed as the mean ± SD. Statistical analysis was performed by ANOVA with Neuman-Keuls test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. WT, wild-type; TG, transgenic; BLM, bleomycin; SAL, saline; ns, not significant.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Expressing, Transgenic Assay, Saline

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Reduced expression of extracellular matrix markers in human MUC5B rs35705950 transgenic mice with lung fibrosis. The relative mRNA expression of extracellular matrix markers was assessed by polymerase-chain reaction and the levels of collagen I was assessed by enzyme immunoassays using commercially available kits following the protocol of the manufacturers. Data are expressed as the mean ± SD. Statistical analysis was performed by ANOVA with Neuman-Keuls test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. WT, wild-type; TG, transgenic; BLM, bleomycin; SAL, saline; ns, not significant.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Expressing, Transgenic Assay, Polymerase Chain Reaction, Enzyme Immunoassay, Saline

Journal: Cells

Article Title: Bleomycin-Induced Pulmonary Fibrosis in Transgenic Mice Carrying the Human MUC5B rs35705950 Variant

doi: 10.3390/cells13181523

Figure Lengend Snippet: Reduced lung fibrosis in human MUC5B rs35705950 transgenic mice. Ashcroft scoring was performed in lung tissue stained with hematoxylin & eosin by blinded experts for the treatment groups. The number of mice in: WT/SAL, n = 8, WT/BLM, n = 18, h-rs35705950-TG/SAL, n = 10, h-rs35705950-TG/BLM, n = 16 ( A , B ). Collagen deposition was evaluated after trichrome staining, and the total lung collagen volume fraction was calculated. The number of mice: WT/SAL, n = 3, WT/BLM, n = 5, h-rs35705950-TG/SAL, n = 3, h-rs35705950-TG/BLM, n = 5 ( C , D ). The lung tissue hydroxyproline content was measured by a colorimetric assay. The number of mice: WT/SAL, n = 8, WT/BLM, n = 18, h-rs35705950-TG/SAL, n = 10, h-rs35705950-TG/BLM, n = 16 ( E ). Scale bars indicate 200 µm. Data are the mean ± S.D. Statistical analysis by ANOVA with Newman-Keuls test. * p < 0.05, ** p < 0.001; **** p < 0.001. WT, wild-type; SAL, saline; BLM, bleomycin.

Article Snippet: The MUC5B staining was performed using a polyclonal anti-MUC5B antibody (Proteintech, Rosemont, IL, USA) that cross-reacts with mouse MUC5b.

Techniques: Transgenic Assay, Staining, Colorimetric Assay, Saline