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: Viruses

Article Title: Broadly Active Antiviral Compounds Disturb Zika Virus Progeny Release Rescuing Virus-Induced Toxicity in Brain Organoids

doi: 10.3390/v13010037

Figure Lengend Snippet: Image-based screening on Zika virus (ZIKV)-infected cells identifies TH3289 and TH6744 as active small molecule inhibitors against ZIKV. ( A ) Experimental workflow of phenotypic antiviral testing of small molecule compounds. Nuclei count was used as an indicator of cytotoxicity, viral antibody staining was quantified to assess the percentage of virus infected cells and virus titer was quantified by end-point dilution assay. ( B ) U87 cells were infected with ZIKV (MOI 10) and treated with structural analogs of TH3289 at 10 µM concentration for 48 h. Infected cells were stained for DAPI and ZIKV NS1 and analyzed by high-throughput imaging. Virus titers from supernatants were determined by end-point dilution assay and inhibition of viral titer was calculated relative to dimethyl sulfoxide (DMSO). Data are presented as a mean of two technical replicates per compound performed in n = 1 biological replicate. Representative images from ZIKV-infected U87 cells treated with DMSO or TH3289 (upper image panels; Scale bar equals 100 µm) and corresponding example of titrated ZIKV from the end-point dilution assay (lower image panels) are shown. DAPI in grey, ZIKV NS-1 in magenta. ( C ) The inhibition of viral titers of Hazara virus (HAZV) and ZIKV from both assays was correlated. In the HAZV assay, SW13 cells were infected with HAZV (MOI 10) and treated with 10 μM of compounds from the in-house library for 24 h. Virus titer from supernatant was determined by end-point dilution assay. Cells were stained for DAPI (in blue) and HAZV NP (in green) and analyzed by high-throughput imaging. Data are presented as a mean of two technical replicates per compound performed in n = 1 biological replicate. ( D ) Chemical structures of top hits from ZIKV and HAZV antiviral screenings and their respective inhibition on viral titers.

Article Snippet: For supernatants, the obtained Ct values were converted to the number of ZIKV RNA molecules using a standard curve that was created using Quantitative Synthetic ZIKV RNA from ATCC (ATCC ® VR-3252SDTM).

Techniques: Virus, Infection, Staining, End-point Dilution Assay, Concentration Assay, High Throughput Screening Assay, Imaging, Inhibition

Journal: Viruses

Article Title: Broadly Active Antiviral Compounds Disturb Zika Virus Progeny Release Rescuing Virus-Induced Toxicity in Brain Organoids

doi: 10.3390/v13010037

Figure Lengend Snippet: Series of antiviral compounds have a therapeutic window and rescue ZIKV-induced CPE in cellular models. ( A – E ) U87 cells were infected with ZIKV (MOI 1) and treated with increasing doses of ( A ) TH3289, ( B ) TH5264, ( C ) TH6744, or ( D ) TH5487 for 48 h. Cell viability was determined by nuclei count (in blue), infected cells by ZIKV NS1 staining (in orange) and virus titer by an end-point dilution assay (in black). Data are presented as mean ± SD from n = 3 independent replicates. ( E ) Curve fitting was performed to calculate EC 50 values for living cells, infected cells and viral titer. ( F ) Vero cells were infected with ZIKV (MOI 10) and treated with 12.5 μM TH6744 or DMSO for 48 h. Cells were stained for ZIKV NS1 (in green), ZIKV Capsid (in red) and DAPI (in blue). Images of n = 1 biological replicate. In the overview image, scale bar equals 100 μm and in the close-up 25 μm. ( G ) U87 cells were infected with ZIKV (MOI 1) and treated with 10 μM TH6744 or DMSO for 24, 48, or 72 h. Cells were stained for Pan-Flavi (in green), Golgi marker GM130 (in red), ER marker KDEL (in yellow), and DAPI (in blue). Images of n = 1 biological replicate. Scale bar equals 50 μm. ( H ) Vero cells were infected with ZIKV (MOI 0.1) and treated with increasing doses of TH6744 for 24 h. Viral titer was measured from the supernatants by end-point dilution assay on Vero cells and the development of CPE were monitored by visual assessment. Data presented as mean ± SD from n = 2 biological replicates.

Article Snippet: For supernatants, the obtained Ct values were converted to the number of ZIKV RNA molecules using a standard curve that was created using Quantitative Synthetic ZIKV RNA from ATCC (ATCC ® VR-3252SDTM).

Techniques: Infection, Staining, Virus, End-point Dilution Assay, Marker

Journal: Viruses

Article Title: Broadly Active Antiviral Compounds Disturb Zika Virus Progeny Release Rescuing Virus-Induced Toxicity in Brain Organoids

doi: 10.3390/v13010037

Figure Lengend Snippet: TH6744 and TH5487 treatment reverses ZIKV-induced neurotoxicity and limits ZIKV propagation in three-dimensional (3D) cerebral organoids. ( A ) Experimental workflow of ZIKV infection on brain organoid model. ( B – F ) Organoids were infected with ZIKV (2 × 10 4 particles/organoid) followed by treatment with 5 μM TH6744, 5 μM TH5487 or DMSO. ( B ) Organoids were dissociated to single cells at 7 or 10 dpi and cell viability was measured by Resazurin assay. Viability is shown relative to uninfected DMSO-treated control at respective timepoints. One datapoint represents one organoid. Data are presented as a mean ± SD of n = 2 biological replicates. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparison analysis. * p < 0.05, ** p < 0.01, ( C ) ZIKV-infected and uninfected whole organoids treated with TH6744 or DMSO for 7 or 10 dpi, stained by Hoechst and imaged by high-content confocal microscopy. Representative images of n = 2 biological replicates per condition. The scale bar equals 500 μm. Hoechst in blue. ( D ) ZIKV-infected organoids that were treated with TH6744 or DMSO were fixed at 7 dpi, cryosectioned and stained with Hoechst and ZIKV NS1 protein antibody. ZIKV NS1 intensity was quantified using Cellprofiler software. Data are presented as single values and mean ± SD from n = 2 biological replicates. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparison analysis. * p < 0.05, ** p < 0.01. ( E ) ZIKV-infected organoids treated with TH6744 or DMSO were fixed at 7 dpi, cryosectioned, stained with Hoechst and ZIKV NS1 protein antibody and imaged by confocal microscopy. Representative images of n = 2 biological replicates per condition. Scale bar equals 500 μm in overview image and 100 μm in close-up. ZIKV NS1 protein in red, Hoechst in blue. ( F ) Viral titer from ZIKV-infected and compound treated organoids at indicated time points was determined by an end-point dilution assay. Viral titer is presented as relative to ZIKV-infected DMSO-treated control at respective timepoints. Data are presented as a mean ± SD from n = 2 biological replicates. Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparison analysis. * p < 0.05, ** p < 0.01.

Article Snippet: For supernatants, the obtained Ct values were converted to the number of ZIKV RNA molecules using a standard curve that was created using Quantitative Synthetic ZIKV RNA from ATCC (ATCC ® VR-3252SDTM).

Techniques: Infection, Resazurin Assay, Control, Comparison, Staining, Confocal Microscopy, Software, End-point Dilution Assay

Journal: Viruses

Article Title: Broadly Active Antiviral Compounds Disturb Zika Virus Progeny Release Rescuing Virus-Induced Toxicity in Brain Organoids

doi: 10.3390/v13010037

Figure Lengend Snippet: TH6744 disturbs late replication cycle steps in ZIKV life cycle. ( A – D ) U87 cells were infected with ZIKV (MOI 1) and treated with indicated compounds for 24, 48 or 72 h. ( A ) ZIKV RNA levels inside the cells were quantified by qPCR. ( B ) Number of ZIKV-infected cells was quantified by virus infectivity assay. ( C ) ZIKV RNA levels in the supernatant were quantified by one-step qRT-PCR. ( D ) ZIKV titers were quantified by end-point dilution assay. ( E ) Schematic overview of time-of-addition experimental setup in U87 cells. ( F ) U87 cells were infected with ZIKV (MOI 10) for a total of 24 h and treated with DMSO control, 10 μM TH6744 or 100 μM Ribavirin for indicated periods during early (entry), late (budding) or throughout replication cycle (post-inoculation). ZIKV titers were quantified by end-point dilution assay. ( G ) Schematic overview of budding experimental setup. ( H ) U87 cells were infected with ZIKV (MOI 10) for 22 h and treated with indicated TH6744 doses for 2 h. Intracellular ZIKV particles were obtained by mechanical cell lysis and extracellular ZIKV particles from the supernatants and both were quantified by end-point dilution assay. ( I ) Average fractions of intracellular and extracellular infectious ZIKV particles of the total particles quantified in ( H ). ( A – D , F , H ) Data are expressed as a mean ± SD from at least n = 3 biological replicates. Statistical significance was determined by using one-way ANOVA with Dunnett’s multiple comparison analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For supernatants, the obtained Ct values were converted to the number of ZIKV RNA molecules using a standard curve that was created using Quantitative Synthetic ZIKV RNA from ATCC (ATCC ® VR-3252SDTM).

Techniques: Infection, Virus, Quantitative RT-PCR, End-point Dilution Assay, Control, Lysis, Comparison

Journal: BMC Cancer

Article Title: Regulation of cancer stem cells by CXCL1, a chemokine whose secretion is controlled by MCM2

doi: 10.1186/s12885-024-12085-0

Figure Lengend Snippet: MCM2 Expression in Cancer Stem Cells and Prognostic Implications. ( A ) Comparison of RNA expression levels of CSC markers and MCM2 in A549 cells cultured in conditioned medium (CM). GAPDH was used as a loading control. ( B ) Comparison of protein expression of CSC markers and MCM2 in A549 cells cultured with CM. β-actin was used as a loading control. Full-length blots are presented in Supplementary Fig. A. ( C ) Comparison of expression levels of CSC marker protein and MCM2 using immunocytochemistry (ICC). ( D ) Kaplan-Meier survival curves for lung cancer patients according to MCM2 gene expression levels. Sample derive from the same experiment and that gels/blots were processed in parallel

Article Snippet: The A549 (CCL-185TM) human lung cacer cell line was obtained from the ATCC and maintained in RPMI 1640 MEDIUM (Cat. No. SH30027.01; Hyclone, Cytiva, USA).

Techniques: Expressing, Comparison, RNA Expression, Cell Culture, Control, Marker, Immunocytochemistry, Gene Expression

Journal: BMC Cancer

Article Title: Regulation of cancer stem cells by CXCL1, a chemokine whose secretion is controlled by MCM2

doi: 10.1186/s12885-024-12085-0

Figure Lengend Snippet: Impact of MCM2 Expression on Characteristics of LCSCs. ( A ) Comparative analysis of differences in sphere formation ability according to MCM2 expression. Comparison of sphere formation after 96 h of treatment of A549 cells with CM. ( B ) Single cell analysis of lung cancer stem cells (LCSCs) based on MCM2 expression. A549 cells were cultured in a 96-well plate to assess sphere-forming ability over a period of 10 days. ( C ) Limiting dilution assay to analyze LCSCs ability. Varying numbers of cells (1, 50, 100, 150, and 200) were plated in each well of a 96-well plate to compare sphere formation capacity. ( D ) Analysis of the expression amount of CSC marker proteins (CD44, ALDH1A1, ALDH1A3) according to MCM2 expression. Full-length blots are presented in Supplementary Fig. B. ( E ) Confirmation of the expression level of CSC regulatory proteins (SOX2, Oct-4, Nanog) according to MCM2 expression. Full-length blots are presented in Supplementary Fig. C. ( F ) Comparative analysis of the expression level of CSC marker proteins by ICC. ( G ) Colony formation assay to measure radioresistance based on MCM2 expression. To this end, 1 × 10 3 cells were planted in each cell and irradiated with 3 Gy of radiation the next day. Sample derive from the same experiment and that blots were processed in parallel. Data represent the mean ± standard deviation of three replicates. Scale bar, 50 μm. *p˂0.005, **p˂0.05

Article Snippet: The A549 (CCL-185TM) human lung cacer cell line was obtained from the ATCC and maintained in RPMI 1640 MEDIUM (Cat. No. SH30027.01; Hyclone, Cytiva, USA).

Techniques: Expressing, Comparison, Single-cell Analysis, Cell Culture, Limiting Dilution Assay, Marker, Colony Assay, Irradiation, 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 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