Journal: Mucosal immunology

Article Title: Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation

doi: 10.1038/mi.2014.109

Figure Lengend Snippet: In A–D , shown is quantitative RT-PCR analysis of NTRK1 and CCL26 transcription in TE-7 cells, EPC2 epithelial cell ALI culture, primary esophageal epithelial cells, and human bronchial epithelial cells. Cells were treated with IL-13 at 100 ng/ml for the indicated periods of time or for 6 days for EPC2 cells. The inset in C shows Western blot for NTRK1 in two independent cultures of primary esophageal epithelial cells stimulated with IL-13. In E , the effect of STAT6 gene silencing by shRNA on NTRK1 and CCL26 induction in TE-7 cells was quantified by RT-PCR. TE-7 cells were stimulated with IL-13 (1 ng/ml) for the indicated periods of time. shCtrl, control shRNA; shSTAT6, shRNA against STAT6 . Data for 3 to 4 independent experiments are presented as mean values for gene expression normalized to the level of GAPDH with standard error measurements.

Article Snippet: Normal human primary bronchial epithelial cells were purchased from Lonza (Lonza, MD, CC-2540) and cultured as previously described .

Techniques: Quantitative RT-PCR, Western Blot, shRNA, Reverse Transcription Polymerase Chain Reaction, Control, Gene Expression

Journal: Mucosal immunology

Article Title: Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation

doi: 10.1038/mi.2014.109

Figure Lengend Snippet: In A , shown is a Western blot analysis of primary esophageal epithelial cells pre-treated with IL-13 for 24 hr and then treated with recombinant human NGF for 0, 5, or 15 min. pNTRK1 indicates phosphorylated protein (arrow). In B , shown is Western blot analysis of TE-7 cells pre-treated with IL-13 for 24 hr and then treated with NGF for 5 min. pNTRK1 and pERK1/2 indicate phosphorylated proteins, arrow points at pNTRK1. In C , the kinetics of NTRK1 phosphorylation were assessed by Western blot. Cells were pre-treated with IL-13 for 24 hr and then treated with NGF for 5, 15 or 30 min. For A–C , phosphorylation was assessed at tyrosine residues Tyr674/675 in the catalytic domain of NTRK1. In D , kinetic analysis of EGR1 and EGR3 mRNA in TE-7 cells pre-treated with IL-13 for 24 hr followed by treatment with NGF for 1, 2, or 6 hrs was performed by RT-PCR. Fold change indicates increase over untreated (no IL-13) cells stimulated with NGF (+NGF). NGF was used at the concentration of 100 ng/ml. Data for 3 independent experiments are presented as mean value with standard error measurements; ****p < 0.0001, *p < 0.05. In E , EGR1 and EGR3 protein levels in TE-7 cells pre-treated with IL-13 for 24 hr followed by treatment with NGF were analyzed by Western blot; p38 serves as a loading control.

Article Snippet: Normal human primary bronchial epithelial cells were purchased from Lonza (Lonza, MD, CC-2540) and cultured as previously described .

Techniques: Western Blot, Recombinant, Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Control

Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 1. Effects of AP on mitochondrial ROS production. (A) Protease (50 μg) activity assessed using a commercial protease activity kit. Trypsin (5 U) was used positive control. (B) Measurement of endotoxin level of AP and inactive AP. Escherichia coli endotoxin was used as positive control. (C) AP and inactive AP-induced mitochondrial ROS production evaluated in primary epithelial bronchial cells using confocal microscopy. (D) AP-induced mitochondrial ROS production sequentially evaluated using confocal microscopy. (E) Decrease in mitochondrial ROS production upon Mito-TEMPO treatment for 1 h evaluated in primary epithelial bronchial cells using confocal microscopy (magnification: 150 × , scale bar: 20 μm). (F) The effect of Mito-TEMPO on protease activity of AP was measured. Data from two independent experiments are shown as the mean ± standard error mean (SEM). *P < 0.05, **P < 0.01 vs. Inactive AP stimulation or endotoxin group.

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activity Assay, Positive Control, Confocal Microscopy

Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 2. Effects of AP on gene expression in primary bronchial epithelial cells. (A) A total of 4115 genes showing an expression change of at least 0.1 standard deviation following AP treatment were selected and clustered according to the expression level. Red and green color represent high and low expression levels, respectively, as shown in the scale bar. (B) Pathways were clustered according to the activity level calculated from the expression level of the genes included in each pathway. Red and green color represent high and low activity levels, respectively, as shown in the scale bar. The functional categories of each pathway are color- coded as shown on the side and below. In the right panel, the average activity levels of the pathways included in each functional category were compared between the two samples (1 h and 24 h after AP treatment). (C) Similarity between samples was measured according to the distribution of pathway activity. Correlation coef ficient values are displayed in color as indicated on the scale bar. (D) Distribution of the number of core differential genes isolated from each sample is shown as a Venn diagram. The ratio of common genes (dot plot) between two samples according to the number (bar plot) of core differential genes is also displayed in the lower plot. (E) GO terms enriched (FDR<0.01) in the core differential genes isolated from AP-treated samples are represented as network graph (upper panel) and tree map plot (lower panel). Each node represents a GO term, and the node size is proportional to the number of genes associated with the GO term. Closely related GO terms are indicated in the same color. Representative GO terms are indicated. (F) Relationship between AP-treated samples were measured using semantic similarity of enriched GO terms (upper panel) from the core differential genes and the distance distribution (middle panel) between core differential genes. In the upper panel, the intensity of red is proportional to the similarity between samples, as shown in the scale bar. In the middle panel, red and green color represent high and low similarity between samples in distance distribution, respectively, as shown in a time-dependent manner in the scale bar. The lower panel schematically shows the distances between samples on the network. Each sample is represented by a node. The size of the node indicates the number of core differential genes. The thicker the edge, the closer the distance between samples. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Gene Expression, Expressing, Standard Deviation, Activity Assay, Functional Assay, Isolation

Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 4. Effects of AP-induced mitochondrial ROS production on epithelial cell apoptosis and epithelial permeability. (A) The change in the expression level of genes included in the apoptosis pathway according to the AP treatment is displayed as a box plot. Activity level of apoptosis pathway according to the AP treatment is also shown. (B) The distances between core differential genes induced by AP treatment and apoptosis-related genes or mitochondrial electron transport-related genes were measured in the network. (C) P53, BCL2, BAX, and active caspase-3 expression after 23 h of incubation following AP stimulation for 1 h was analyzed using western blotting (representative image). β-Actin was used as a loading control. (D) JC-1 staining for mitochondrial membrane potential (ΔΨm) on primary human bronchial epithelial cells under confocal microscopy (magnification: 60 × , scale bar: 20 μm). (E) Changes in the activity of pathways related to the cell adhesion function according to AP treatment time were measured. Data are presented as mean ± SEM. The pathways studied were the adherens junction, tight junction, gap junction, and actin cytoskeletal pathways. (F) After core differential genes in the GO terms and pathways related to the cell adhesion function were selected, the average expression level of the neighboring genes of each core differential gene was measured, and their distribution was observed as a box plot. *P < 0.05, **P < 0.01 vs. 1 h after AP stimulation group. (G) ZO-1, claudin 1, occludin, and E-cadherin expression after 23 h of incubation following AP stimulation for 1 h. (H) Monolayer permeability of primary bronchial epithelial cells is expressed as a percentage of change in FITC-dextran (Mw = 20,000 Da) fluorescence intensity following 23 h of incubation following AP stimulation for 1 h. Data from three experiments are shown as mean ± SEM. *P < 0.05, **P < 0.01 vs. control group (no AP stimulation or Mito-TEMPO pretreatment); #P < 0.05, ##P < 0.01 vs. AP stimulation group without Mito-TEMPO.

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Permeability, Expressing, Activity Assay, Incubation, Western Blot, Control, Staining, Membrane, Confocal Microscopy, Fluorescence

Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 5. Effects of Mito-TEMPO on AP-induced changes in gene expression in primary bronchial epithelial cells. (A) In the left panel, 3742 genes showing an expression change of at least 0.1 standard deviation after AP and Mito-TEMPO treatment were selected and clustered according to the expression level. Red and green color represent high and low expression levels, respectively, as shown in the scale bar. In the right panel, the gene expression change caused by Mito-TEMPO was compared in parallel with that in the AP alone treatment group. (B) In the left panel, distribution of the number of core differential genes isolated from each sample is shown as a Venn diagram. Enriched GO terms (FDR <0.01) in core differential genes isolated from AP and Mito-TEMPO-treated samples are represented as a network graph (right panel) and dot plot (lower panel). (C) In the left panel, core differential genes selected from both AP and Mito-TEMPO treated samples were compared with those selected from AP alone-treated samples. The number of common genes between AP and Mito-TEMPO-treated samples and AP alone-treated samples is indicated in red. In the right panel, the functional association of these common genes is indicated by dots. (D) Locations of the selected core differential genes in AP alone-treated samples and in AP and Mito-TEMPO-treated samples (1 h) are displayed in the interaction network. For each sample, the size of the major node cluster composed of core differential genes and the average distance between nodes within the cluster are shown in the panel below. (E) Pathways were clustered according to the activity level calculated from the expression level of the genes included in each pathway. The functional categories of each pathway are color-coded as shown on the side and below. In the right panel, the pathway activity change caused by Mito-TEMPO was compared in parallel with that in the AP alone treatment group. The lower panel shows a correlation plot between the AP-only samples and the AP and Mito-TEMPO-treated samples. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Gene Expression, Expressing, Standard Deviation, Isolation, Functional Assay, Activity Assay

Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 6. Comparative analysis with external datasets of hypoxia-treated bronchial epithelial cells. (A) Core differential genes were selected from two external datasets (GSE68378 and GSE121773) of bronchial epithelial cells exposed to hypoxia or treated with reactive oxygen species, and their distributions were plotted as Venn diagrams. (B) In the left and middle panels, enriched pathways and GO terms (FDR <0.001) associated with the core differential genes, respectively, were compared between samples. As shown in the scale bar, the significance increased as the black color increased. In the right panel, relationships between samples were measured using semantic similarity of the enriched GO terms from the core differential genes. Red color intensity is proportional to the similarity between samples, as shown in the scale bar. (C) The pattern of distance distribution in the network between core differential genes was used to measure similarity between samples. Red and green color represent high and low similarity between samples in distance distribution, respectively, as shown in the scale bar. (D) In the left panel, pathways were clustered according to the activity level calculated from the expression level of the genes included in each pathway. The functional categories of each pathway are color-coded on the side. In the middle panel, similarity between samples was measured according to the distribution of pathway activity. Correlation coefficient values are displayed in color as indicated on the scale bar. The right panel shows correlation plots between AP-treated samples (1 and 24 h) and selected external samples. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activity Assay, Expressing, Functional Assay

Journal: Biomaterials advances

Article Title: Fabrication of heparinized small diameter TPU/PCL bi-layered artificial blood vessels and in vivo assessment in a rabbit carotid artery replacement model.

doi: 10.1016/j.msec.2021.112628

Figure Lengend Snippet: Fig. 7. Analysis of connectivity map data. (A) A total of 5170 genes showing an expression change of at least 0.1 standard deviation following treatment with five uncoupling agents (CCCP, FCCP, bongkrek acid, isorotenone, and elesclomol) were selected and clustered according to the expression level. Red and green color represent high and low expression levels, respectively, as shown in the scale bar. (B) The ratio of common core differential genes, which was adjusted for the total number of core differential genes, was compared between the connectivity map samples and the AP-treated samples. Black color intensity is proportional to the adjusted ratio of common genes between samples, as shown in the scale bar. In the right panel, the distribution of adjusted common core differential gene numbers is displayed for each individual connectivity map sample. (C) Correlation between pathway activity of individual samples in the connectivity map and that of AP- treated samples was measured. (D) Samples treated only with CCCP or FCCP were clustered according to the pathway activity level. The functional categories of each pathway are color-coded as shown on the side and below. The right panel shows a correlation plot between AP-treated samples (1 and 18 h) and CCCP- or FCCP- treated samples. (E) FCCP-induced mitochondrial ROS production was sequentially evaluated in primary epithelial bronchial cells by confocal microscopy (magnification: 150 × , scale bar: 20 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Human primary bronchial epithelial cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Standard Deviation, Activity Assay, Functional Assay, Confocal Microscopy

Journal: bioRxiv

Article Title: A novel isoform of ACE2 is expressed in human nasal and bronchial respiratory epithelia and is upregulated in response to RNA respiratory virus infection

doi: 10.1101/2020.07.31.230870

Figure Lengend Snippet: 3a. Agarose gel electrophoresis image of transcript-specific ACE2 RT-PCRs from different cell types studied. Size standard = NEB Low Molecular Weight ladder. 3b. RT-qPCR analysis of transcript-specific PCRs for long and short ACE2 expression in cell lines and airway cells. Analysis was done at least in duplicate and from different passages or donors in all lines but RPE-1, Caco-2 and HEK 293 cells where analysis was done in duplicate from one passage. 3c. Graphs showing relative expression of short ACE2 transcript and long ACE2 transcript in nasal epithelial cells at different stages of differentiation at air-liquid interface (Day 1, 4, 8, 14, week 4, week 9, week 12) (n=3 for each time point except week 12 where n=2) and primary nasal brushings (n=6). Scale = reads mapped to exon/exon boundary per million mapped reads. Error bars = standard error of the mean. 3d. Graphs showing relative expression of short ACE2 transcript and long ACE2 transcript in nasal (n=11) and bronchial (n=11) ALI cultures from healthy donors, as determined using transcript specific qPCR. Data were analysed using Mann Whitney U test. 3e. Agarose gel electrophoresis image of transcript-specific PCR products multiple cell types on multiple tissue control panel. This shows expression of the long transcript of ACE2 in heart, placenta lung, liver skeletal muscle, kidney and pancreas but expression of the short transcript of ACE2 only in lung, liver and kidney. 3f. Graphs showing relative expression of short ACE2 , long ACE2 and total ACE2 transcript in a Multiple Tissue cDNA panel 1 (636742, Takara), as determined using transcript-specific ACE2 RT-qPCR. n.d.= not detected. Analysis was done in duplicate runs on the same day, n=2 in different days.

Article Snippet: Primary bronchial epithelial cells were expanded in Airway Epithelial Cell Growth medium (Promocell) up to passage 1 as previously described .

Techniques: Agarose Gel Electrophoresis, Molecular Weight, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: A novel isoform of ACE2 is expressed in human nasal and bronchial respiratory epithelia and is upregulated in response to RNA respiratory virus infection

doi: 10.1101/2020.07.31.230870

Figure Lengend Snippet: 4a. Schematic illustration of predicted long and short protein isoforms of ACE2 and position of antigen sequences used to generate antibodies used. 4b. Left panel: Representative western blot of Vero cells and nasal cells (n=3) grown at ALI blotted with ACE2 antibody raised to C-terminal domain (amino acids 788-805). Grey arrow points to glycosylated long ACE2 detected in both Vero E6 and nasal ALIs, black arrow points to unglycosylated long ACE2 in nasal ALI, red arrow points to unglycosylated short ACE2 in nasal ALI. Middle panel: Representative western blot of Vero cells and nasal cells grown at ALI blotted (n=3) with ACE2 antibody raised to N-terminal domain (amino acids 200-300). Grey arrow points to glycosylated long ACE2 detected in both cell types. Right panel: Western blot of bronchial cells grown at ALI blotted with ACE2 antibody raised to epitopes across the protein (amino acids 18-740). 4c. Representative IF confocal images of ALI differentiated primary bronchial epithelial cells stained with anti-alpha tubulin (green), Acti-Stain 555 Phalloidin (grey), DAPI (blue) and ACE2 (red) detected with C-terminal domain antibody (top panel) or antibody detecting epitopes across the protein (bottom panel). N=2 4d. Schematic illustration of deciliation protocol using calcium shock (left) and western blot of whole and deciliated BCi-NS1.1 cells, deciliation wash, and cilia pellet (right). Red arrow points to short ACE2 which not enriched on cilia relative to long ACE2 enrichment. The graph shows semi-quantitative analysis of the Western blots by densitometric analysis (n=4).

Article Snippet: Primary bronchial epithelial cells were expanded in Airway Epithelial Cell Growth medium (Promocell) up to passage 1 as previously described .

Techniques: Western Blot, Staining

Journal: bioRxiv

Article Title: A novel isoform of ACE2 is expressed in human nasal and bronchial respiratory epithelia and is upregulated in response to RNA respiratory virus infection

doi: 10.1101/2020.07.31.230870

Figure Lengend Snippet: 6a. Undifferentiated primary bronchial epithelial cell (PBEC) monolayer cultures (N=3) (top) or in vitro differentiated (ALI) PBEC cultures (N=3) (bottom) were treated with IFN-beta (100 or 1000 IU/ml) for 24h and ACE2 transcripts (left panel) and induction of IFN-response genes (MX1 and CXCL10) (right panel) were measured by RT-qPCR. Data were analysed using Students t-test. 6b. in vitro differentiated (ALI) nasal epithelia cells (NEC) (n=11) (left panel) or in vitro differentiated (ALI) bronchial epithelia cells (BEC) (n=11) (right panel) were infected with rhinovirus (RV16) (MOI of 1) or mock-infected using a UV-irradiated control (UV-RV16). Nasal cells were collected from 3 female, 8 male patients with a mean age of 45.31+/-3.23 (SEM). After 24h, induction of ACE2 isoform expression was assessed by RT-qPCR with transcript-specific primers. Data were analysed using non-parametric Wilcoxon test. 6c. BCi-NS1.1 cells were grown at ALI and then infected on the apical side for 1 hour with 100,000 pfu of SARS-Cov-2 strain nCoV/Victoria/1/2020 obtained from Public Health England (PHE), UK. Cells were harvested in QIAzol at 1h post infection and at 72h, RNA extracted and quantitative RT-qPCR performed to detect SARS-CoV-2 using 2019-nCoV_N1 primers and the housekeeping genes HPRT, 18S and RNAse P using the dCt method. 1 hour and 72 hours after infection induction of ACE2 transcript expression was assessed by RT-qPCR with transcript-specific primers (left). SARS-CoV-1 infection was confirmed by CoV-N1 RT-qPCR 1 and 72 hours after infection (right). Data were analysed using non-parametric Wilcoxon test. N=4.

Article Snippet: Primary bronchial epithelial cells were expanded in Airway Epithelial Cell Growth medium (Promocell) up to passage 1 as previously described .

Techniques: In Vitro, Quantitative RT-PCR, Infection, Irradiation, Expressing

Journal: bioRxiv

Article Title: A novel isoform of ACE2 is expressed in human nasal and bronchial respiratory epithelia and is upregulated in response to RNA respiratory virus infection

doi: 10.1101/2020.07.31.230870

Figure Lengend Snippet: 7a. Bronchial epithelial brushes from healthy controls (n=13) or severe asthmatic (n=11) donors were harvested and RNA extracted for analysis of ACE2 transcript expression by transcript-specific RT-qPCR. Data were analysed using nonparametric Mann-Whitney test. 7b. Bronchial epithelial cells from healthy (n=11) or severe asthmatic (n=7) donors were grown at ALI and infected with rhinovirus (RV16) (MOI of 1) or mock-infected using a UV-irradiated control. After 24h, induction of ACE2 transcripts was assessed by RT-qPCR with transcript-specific primers in duplicate. The fold-induction of each transcript by RV16 was quantified using the ddCt method using UV-RV exposed cells as control. Activation of anti-viral response in RV16-infected ALI cultures was demonstrated by detection of IL29/IL28 in basolateral supernatants by ELISA (healthy n=14, severe n=8). Data were analysed using Student’s t-test.

Article Snippet: Primary bronchial epithelial cells were expanded in Airway Epithelial Cell Growth medium (Promocell) up to passage 1 as previously described .

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Infection, Irradiation, Activation Assay, Enzyme-linked Immunosorbent Assay