Journal: Stem cell reviews

Article Title: Endothelial Cell Mediated Promotion of Ciliated Cell Differentiation of Human Airway Basal Cells via Insulin and Insulin-like Growth Factor 1 Receptor Mediated Signaling

doi: 10.1007/s12015-016-9707-z

Figure Lengend Snippet: Establishment of basal cell (BC) and endothelial cell (EC) co-culture system to study BC differentiation into a mucociliated epithelium. A. Immunofluorescence assessment of a normal human airway biopsy with staining for BC (KRT5, green), EC (CD31, red) and nuclei (DAPI, blue). Scale bar 50 μm. B. Schematic representation of the BC and EC co-culture system to study the impact of EC on BC differentiation into a mucociliated epithelium. C. Immunofluorescence assessment of BC and EC co-culture with staining for BC (KRT5, red), EC (VE-cadherin, green) and nuclei (DAPI, blue). Upper panel, BC cultured alone. Lower panel, BC and EC co-culture. White dashed line outlines the membrane of the Transwell insert. Scale bar 20 μm. D. Immunofluorescence staining of the epithelial layer of ALI day 28 cultures. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio and then stained for ciliated cells (β-tubulin IV, green), secretory cells (SCGB1A1, red) and nuclei (DAPI, blue). Scale bar 20 μm. E. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio on air-liquid interface (ALI) culture for 7 days to assess the impact of EC on the early stages of BC differentiation into a mucociliated epithelium at the molecular level. TaqMan PCR analysis to assess mRNA expression of BC (KRT5), intermediate (KRT8), secretory (non-mucus producing, SCGB1A1 and mucus producing, MUC5B) and ciliated cell (FOXJ1) markers in BC cultured alone or co-cultured with EC cells at 10:1 and 2:1 (BC:EC) ratio. Bars indicate the mean for n=6 independent experiments each performed in triplicate and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC alone. The experiments for E were performed with n=4 independent donors of BC and EC.

Article Snippet: The samples were then stained with the following primary antibodies: rabbit monoclonal CD31 (5 μg/ml; ab76533; Abcam, Cambridge, UK); goat polyclonal VE-Cadherin (1 μg/ml; AF938; R&D Systems Inc., Minneapolis, MN), mouse monoclonal KRT5 (4 μg/ml; MA5-12596; ThermoFisher Scientific); rabbit polyclonal KRT5 (2 μg/ml; PA1-37974; ThermoFisher Scientific); rabbit polyclonal KRT8 (10 μg/ml; NBP2-16094; Novus Biological, Littleton, CO); rabbit polyclonal SCGB1A1 (5 μg/ml; RD181022220; BioVendor LLC, Candler, NC); rabbit polyclonal MUC5B (4 μg/ml; sc-20119; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal β-tubulin IV (10 μg/ml; MU178-UC; Biogenex, Fremont, CA) at 4°C overnight.

Techniques: Co-Culture Assay, Immunofluorescence, Staining, Cell Culture, Expressing

Journal: Stem cell reviews

Article Title: Endothelial Cell Mediated Promotion of Ciliated Cell Differentiation of Human Airway Basal Cells via Insulin and Insulin-like Growth Factor 1 Receptor Mediated Signaling

doi: 10.1007/s12015-016-9707-z

Figure Lengend Snippet: Endothelial cell (EC) co-culture increases differentiation of basal cells (BC) into ciliated cells. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio on air-liquid interface (ALI) culture for 28 days to assess the impact of EC on BC differentiation into a mucociliated epithelium at the histological level via immunofluorescence staining with cell type specific markers. A. KRT5+ BC. Sections of cells were stained for KRT5 (red) and DAPI (nuclei, blue). B. KRT8+ intermediate cells. Sections of cells were stained for KRT8 (red) and DAPI (nuclei, blue). C. SCGB1A1+ secretory cells. Sections of cells were stained for SCGB1A1 (red) and DAPI (nuclei, blue). D. MUC5B+ secretory cells. Sections of cells were stained for MUC5B (red) and DAPI (nuclei, blue). E β-tubulin IV+ cells. Sections of cells were stained for β-tubulin IV (ciliated, green) and DAPI (nuclei, blue). A–E. Scale bar 20 μm. H. Quantification of KRT5+, KRT8+, SCGB1A1+, MUC5B+ and β-tubulin IV+ cells. The bars indicate the mean for n=4 independent experiments and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC cultured alone. The experiments for A–H were performed with n=4 independent donors of BC and EC.

Article Snippet: The samples were then stained with the following primary antibodies: rabbit monoclonal CD31 (5 μg/ml; ab76533; Abcam, Cambridge, UK); goat polyclonal VE-Cadherin (1 μg/ml; AF938; R&D Systems Inc., Minneapolis, MN), mouse monoclonal KRT5 (4 μg/ml; MA5-12596; ThermoFisher Scientific); rabbit polyclonal KRT5 (2 μg/ml; PA1-37974; ThermoFisher Scientific); rabbit polyclonal KRT8 (10 μg/ml; NBP2-16094; Novus Biological, Littleton, CO); rabbit polyclonal SCGB1A1 (5 μg/ml; RD181022220; BioVendor LLC, Candler, NC); rabbit polyclonal MUC5B (4 μg/ml; sc-20119; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal β-tubulin IV (10 μg/ml; MU178-UC; Biogenex, Fremont, CA) at 4°C overnight.

Techniques: Co-Culture Assay, Cell Culture, Immunofluorescence, Staining

Journal: Stem cell reviews

Article Title: Endothelial Cell Mediated Promotion of Ciliated Cell Differentiation of Human Airway Basal Cells via Insulin and Insulin-like Growth Factor 1 Receptor Mediated Signaling

doi: 10.1007/s12015-016-9707-z

Figure Lengend Snippet: siRNA mediated knockdown of INSR and IGF1R in basal cells (BC) suppresses ciliated cell differentiation. Basal cells with transfected with either control, INSR or IGF1R specific siRNA and cultured on ALI for 7 days to assess the impact of each receptor on the early stages of BC differentiation into a mucociliated epithelium at the molecular level. A. TaqMan PCR analysis to assess INSR mRNA expression at ALI day 0. Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. B. TaqMan PCR analysis to assess IGF1R mRNA expression at ALI day 0. Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. C. TaqMan PCR analysis to assess mRNA expression of BC (KRT5), intermediate (KRT8), secretory (non-mucus producing, SCGB1A1 and mucus producing, MUC5B) and ciliated cell markers (FOXJ1, MYB and DNAI1) in BC at ALI day 7 following siRNA mediated knockdown of INSR (black bars) and IGF1R (grey bars). Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC transfected with control siRNA. The experiments for A–C were performed with n=3 independent donors of BC.

Article Snippet: The samples were then stained with the following primary antibodies: rabbit monoclonal CD31 (5 μg/ml; ab76533; Abcam, Cambridge, UK); goat polyclonal VE-Cadherin (1 μg/ml; AF938; R&D Systems Inc., Minneapolis, MN), mouse monoclonal KRT5 (4 μg/ml; MA5-12596; ThermoFisher Scientific); rabbit polyclonal KRT5 (2 μg/ml; PA1-37974; ThermoFisher Scientific); rabbit polyclonal KRT8 (10 μg/ml; NBP2-16094; Novus Biological, Littleton, CO); rabbit polyclonal SCGB1A1 (5 μg/ml; RD181022220; BioVendor LLC, Candler, NC); rabbit polyclonal MUC5B (4 μg/ml; sc-20119; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal β-tubulin IV (10 μg/ml; MU178-UC; Biogenex, Fremont, CA) at 4°C overnight.

Techniques: Cell Differentiation, Transfection, Cell Culture, Expressing

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Cluster identification. SAE cell types were identified from Dropseq single cell RNA sequencing datasets from six individuals (three healthy nonsmokers and three healthy smokers) using established cell specific markers including KRT5 for basal cells, SCGB1A1 for club cells, MUC5AC for goblet cells, and FOXJ1/DNAI1 for ciliated cells . Club cells were identified by the presence of SCGB1A1, low levels of the basal marker KRT5, and the absence of the differentiated goblet cell marker MUC5AC. Club cells underwent Seurat unsupervised clustering which identified three unique subsets of club cells: club cells 1–3 (CC1, CC2, CC3, respectively). One nonsmoker sample was enriched in pseudogenes ( arrow ); the pseudogene expression data was not used in subsequent analyses. A three-dimensional representation helps to discern the distinct localization of the three clusters in the tSNE plot. The distribution of CC1, 2 and 3 within the original low resolution cluster map of the airway cells is shown in Supplementary Fig. . PNEC pulmonary neuroendocrine cells, APC antigen-presenting cells; NCL high represents a distinct cluster of cells identified in Zuo et al. 2020 . The relative size of the populations shown on the tSNE plot is not representative of the proportions of cells found in the tissue in vivo. The single-cell RNA-seq process involves separation of the epithelium into single, live cells, a process that incurs greater loss and cell death among larger cells such as ciliated cells and favors preservation of smaller cells such as basal cells. It is potentially possible for a cell type or subcluster to be under- or over-represented in this analysis. b Quantification of the average percent of club cells in each club cell subset relative to all club cells. c Heatmap of the top ten differentially expressed genes in each subcluster highlighting the differences among the three subclusters. Yellow represents increased expression levels. For the identity of genes contributing to this heatmap, see Supplementary Fig. . d Representation of each of the club cell subsets in each of the six study subjects. Note the presence of the pseudogene expression in the first nonsmoker subject ( arrow ) that was marked in panel a and excluded from subsequent analysis .

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: RNA Sequencing, Marker, Expressing, In Vivo, Preserving

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Airway in adult normal nonsmoker human lung was analyzed for club cell subtypes. Club cells were identified as SCGB1A1 + cells (green) and subsets were identified using various markers in the single cell sequencing. a Secretoglobin family 3A member 1 (SCGB3A1) (red) and secretory leukocyte protease inhibitor (SLPI) (purple). b SCGB1A1 (green), mucin 5B (MUC5B; purple). Club cells that only express SCGB1A1 but no secondary markers are denoted with a white arrow. Club cells expressing multiple markers are denoted with a white arrowhead. Scale bar = 20 μm.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Sequencing, Protease Inhibitor, Expressing

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Differentially expressed genes in progenitor vs. effector club cells a .

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques:

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Most commonly enriched genes in each club cell subcluster.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Expressing, Binding Assay

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Assessment of epithelial cell differentiation using Slingshot pseudotime value. Shown are epithelial cells differentiating in the secretory pathway including basal cells (BC, pink), intermediate cells (IC, orange), the progenitor club cell cluster (CC1, red), the effector club cell cluster (CC3, yellow), the proliferating club cell cluster (CC2, blue) and mucous cells (MC, green) for nonsmokers (NS) and smokers (S) plotted separately. Superimposed on each tSNE plot, the Slingshot pseudotime analysis shows the path of differentiation assuming basal cells as a starting point. b Pseudotime analysis. The extent of differentiation from the starting point was evaluated using a pseudotime analysis separately for nonsmokers (NS) and smokers (S). The results for each cluster have been plotted to enable pairwise comparisons. Significance of differences in the differentiation of each cluster is indicated by the p value to the right of the graph. p values were determined using a t test applied to the pseudotime values extracted by Slingshot. c Stem-progenitor activity of CC1 correlates with elevated KRT5 expression. Expression levels measured by the number of unique molecular identifiers (UMI) detected per cell during single cell RNA sequencing were plotted for KRT5 and KRT7 as a function of cluster for NS and S. Significant differences between NS and S are indicated by p values. Note that KRT5 levels in CC1 in NS have a mean that is greater than KRT5 expression in IC. KRT5 levels are considered a marker of stem-progenitor cells and are commonly thought to steadily fall as differentiation proceeds (see Supplementary Fig. ). Expression of high levels of KRT5 in CC1 support CC1 as a branch point in the trajectory. At the same time, KRT7, a marker of club cells (see Supplementary Fig. ), is clearly elevated in CC1 confirming the identity of CC1 as club cells rather than basal cells. d Change in branch point in smokers correlates with elevated SPDEF activity. Significant changes in expression levels of genes known to be up- or down-regulated by SPDEF were assessed in CC1 and CC3. Up-regulated genes included CEACAM5 and GCNT3. Down-regulated genes included SCGB1A1, SCGB3A1, LTF, and AQP5. Note that AQP5 is also up-regulated by oxidant stress such as exposure to cigarette smoke, potentially explaining the lack of down-regulation by smoking in CC1 and CC3. Significance of difference for panels c and d was determined based on the combined conditions of an adjusted p value < 0.05 (Bonferroni corrected) and a log fold change > 0.25. Violin plots in panels b – d include box-and-whisker plots showing the mean, 2nd and 3rd quartiles (box), range from minimum to maximum (whiskers), and outliers (dots).

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Cell Differentiation, Activity Assay, Expressing, RNA Sequencing, Marker, Whisker Assay

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Nonsmoker primary small airway epithelium (SAE) basal cells were cultured in air–liquid interface (ALI) culture for 28 days. a Morphology of the cultures at day 7 and 28. Shown are cross sections of the ALI culture, hematoxylin and eosin stain. b , c Multicolor immunofluorescence assessment of club cell subtypes. All club cells were identified using SCGB1A1 marker (green); effector club cells were further identified by the co-expression of MUC5B (panel b red) or SCGB3A1 (panel c red). DAPI identifies the nucleus of all cells. The number of days after establishment of the air liquid interface is noted. Green arrowheads mark cells that are SCGB1A1 + club cells that lack other markers. Orange arrowheads mark cells that are positive for SCGB1A1 and either MUC5B or SCGB3A1. Red arrowheads mark cells that are positive for MUC5B or SCGB3A1, but express little if any SCGB1A1. Scale bars are 50 μm. d Gene expression on ALI over time. TaqMan probes for genes enriched in effector club cells were assessed by qPCR and normalized to an 18S rRNA control. Probes were tested at days 0, 7, 14, and 28 of SAE nonsmoker cells differentiated in ALI. Error bars represent standard deviation among three different ALI cultures. Genes assessed for expression include MUC5B, SCGB1A1, SLPI, PIGR, and LYZ.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Cell Culture, H&E Stain, Immunofluorescence, Marker, Expressing, Gene Expression, Control, Standard Deviation

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Smoking-related reprogramming of small airway epithelium club cells.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques:

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Numbers of club cells in smokers vs. nonsmokers. Club cells from nonsmoker and smoker human small airway epithelium (SAE) cytopreps, identified by SCGB1A1 + immunostaining and absence of KRT5, were quantified and compared against total cells, quantified by DAPI staining, per cytoprep. Three samples of each phenotype were evaluated by a blinded observer, with over 500 total cells per sample; plots show mean ± standard error. b Proportion of club cells exhibiting characteristics of subclusters 1 (progenitor), 2 (proliferative), and 3 (effector) in the population of club cells derived from nonsmokers and smokers. c Effect of cigarette smoke extract (CSE) exposure on differentiation of small airway epithelial club cells. Exposure of basal cells differentiated on air liquid interface (ALI) to cigarette smoke extract (3% Marlboro Red) led to a decrease in defense-related transcript in cells: CYP1A1, positive control demonstrating exposure to cigarette smoke extract; MUC5B; PIGR; SLPI; LYZ; and MUC1. Values are expressed as normalized expression relative to 18S rRNA. Each point represents one well of an experiment done in triplicate; plot shows mean ± standard deviation; p values are from a two-sided unequal variance Student’s t -test. All data is from ALI day 28.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Immunostaining, Staining, Derivative Assay, Positive Control, Expressing, Standard Deviation

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Immunofluorescence analysis of cytopreps containing club cells (SCGB1A1 + ) expressing the effector club cell genes PIGR or MUC5B. White arrowheads indicate dual-labeled cells. White arrows indicate SCGB1A1 + club cells that lack significant expression of a PIGR or MUC5B. Black arrows mark examples of SCGB1A1-negative non-club cells in the cytoprep. Bar = 20 µm. b Total number of dual labeled (SCGB1A1 + PIGR + or SCGB1A1 + MUC5B + ) cells, or SCGB1A1 single labeled cells were quantified by a blinded observer in nonsmokers and compared with smokers. Plot shows mean of three experiments ± standard error; p values are from a two-sided unequal variance Student’s t -test.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Immunofluorescence, Expressing, Labeling

Journal: Respiratory research

Article Title: Modulation of the Epithelial-mesenchymal transition process by Forkhead Box C2 in the repair of airway epithelium after injury.

doi: 10.1186/s12931-025-03150-8

Figure Lengend Snippet: Fig. 3 Knockdown of FOXC2 ameliorates airway epithelial injury in NAPH-treated mice. A Experimental diagram of in vivo experiments. B, C FOXC2 expression in the lung tissues by qPCR and immunoblot analysis. D Immunohistochemistry indicated the expression of FOXC2 in the bronchiolar epithelium of lung tissues. E Morphological observation of the lung tissues at 14 days post-NAPH using hematoxylin & eosin staining. Black arrows indicate injured cells. F Club cells in the lung tissues were stained with an anti-CCSP antibody, and representative immunofluorescence images were displayed. White arrows indicate CCSP-positive cells. Scale bar: 50 μm. DPN: days post NAPH. Data are presented as mean ± SD. **p < 0.01

Article Snippet: The cell slices were then incubated with anti-rabbit IgG secondary antibody (1:200; CST, MA, USA) for 1 h. For double immunofluorescence staining, the lung sections were incubated with primary antibodies anti-CCSP (1:50; Santa Cruz, CA, USA) and anti-vimentin (1:100; Affinity, Changzhou, China) or anti-N-cadherin (1:100; Affinity, Changzhou, China).

Techniques: Knockdown, In Vivo, Expressing, Western Blot, Immunohistochemistry, Staining, Immunofluorescence

Journal: Respiratory research

Article Title: Modulation of the Epithelial-mesenchymal transition process by Forkhead Box C2 in the repair of airway epithelium after injury.

doi: 10.1186/s12931-025-03150-8

Figure Lengend Snippet: Fig. 4 Knockdown of FOXC2 suppresses EMT in NAPH-treated mice. A Immunoblots were performed in the lung tissues for the indicated proteins. B The lung tissues were stained with anti-vimentin and anti-CCSP antibodies, and representative images of dual staining were shown. Yellow arrows indicate vimentin- and CCSP-positive cells. Scale bar: 50 μm. C Representative images of dual immunofluorescence staining of N-cadherin and CCSP in the lung tissues. Yellow arrows indicate N-cadherin- and CCSP-positive cells. Scale bar: 50 μm. Data are presented as mean ± SD. **p < 0.01

Article Snippet: The cell slices were then incubated with anti-rabbit IgG secondary antibody (1:200; CST, MA, USA) for 1 h. For double immunofluorescence staining, the lung sections were incubated with primary antibodies anti-CCSP (1:50; Santa Cruz, CA, USA) and anti-vimentin (1:100; Affinity, Changzhou, China) or anti-N-cadherin (1:100; Affinity, Changzhou, China).

Techniques: Knockdown, Western Blot, Staining, Immunofluorescence

Journal: Scientific reports

Article Title: Synergistic inhibition effects of andrographolide and baicalin on coronavirus mechanisms by downregulation of ACE2 protein level.

doi: 10.1038/s41598-024-54722-5

Figure Lengend Snippet: Figure 1. Staining of pathological tissues of mouse lung tissues ACE2 and CC10. SARS-COV-2: mice infection with a pseudovirus; Drug intervention group: Andrographolide and baicalein intervene in infected mice. The scale bars of 100 × and 400 × magnification in the panels represent 200 μm and 50 μm, respectively. *P < 0.05, **P < 0.01 SARS-COV-2 compared with Drug intervention group.

Article Snippet: Immunohistochemical analysis of paraffin-embedded mouse lung tissue slides was performed using ACE2 (Affinty, AF5165) and CC10 antibody (Proteintech, 10,490–1-AP) under 20 × and 40 × lenses.

Techniques: Staining, Infection