Journal: Allergy

Article Title: Human Pulmonary Neuroendocrine Cells Respond to House Dust Mite Extract With PAR-1 Dependent Release of CGRP.

doi: 10.1111/all.16416

Figure Lengend Snippet: FIGURE 1 | Characterization of iPNEC and ePNEC cultures. (A) Quantitative RT-PCR showing expression of characteristic PNEC markers in human cultured iPSC and -HBEC-derived iPNEC (□ female, 22; ■ male, 32) and ePNEC (● male, 52; ▲ male, 56; ○ female, 55) and control HBEC at day 60 in ALI culture, respectively. (B) Mean fluorescent intensity (MFI) of CHGA+/Hoechst+ cells (left) and SYP+/Hoechst+ cells [34] at day 60. Data is representative of four different culture plate wells per group for one biological donor for each cell type (ePNEC male, 52; iPNEC male, 32). (C) Representative IF images show SYP+ cells and nuclei are counterstained with Hoechst. 20× magnification and scale bars at 100 μm. (D) Single- cell RNA sequencing identifies two ePNEC clusters in 60-day-old ePNEC differentiated cells. Each dot represents one well and data shown for mean ± SEM. Mann–Whitney test performed for (A, B); * < 0.05. ASCL1, Achaete-Scute Family BHLH Transcription Factor 1; CHGA, Chromogranin A; ePNEC, epithelial-derived pulmonary neuroendocrine cells; iPNEC, iPSC-derived pulmonary neuroendocrine cells; iPSC, induced pluripotent stem cells; SYP, Synaptophysin.

Article Snippet: Cells were grown using the bronchial epithelial cell growth kit from ATCC (PCS300- 040).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Derivative Assay, Control, RNA Sequencing, MANN-WHITNEY

Journal: Allergy

Article Title: Human Pulmonary Neuroendocrine Cells Respond to House Dust Mite Extract With PAR-1 Dependent Release of CGRP.

doi: 10.1111/all.16416

Figure Lengend Snippet: FIGURE 4 | CGRP release after HDM stimulation is PAR1-dependent (A) Single-cell transcriptomics data of iPNECs (GSE146990, Hor et al.) and ePNEC for PAR1 (F2R), PAR2 (F2RL1), PAR3 (F2RL2), and PAR4 (F2RL3) genes. (B) SYP and PAR1 immunohistochemical co-staining in naive iPNEC (male, 32). 20× and 100× magnification and scale bars at 50 and 10 μm, respectively. (C, D) CALCB mRNA (C) and CGRP protein (D) expression in different ePNEC (● male, 52; ▲ male, 56; ○ female, 55) conditions after 2 h: 50 μL PBS, HDM (1200 μg/mL), PAR1 inhibitor Vorapaxar (80 μM) and PAR1 agonist TFLLR-NH2 (2 μM). (E) CALCB mRNA RT-PCR expression in different ePNEC conditions after 2 h: 50 μL PBS, HDM (1200 μg/mL), HDM co-incubation with protease inhibitors Chymostatin (broad spectrum, 10 μg/mL), PMSF (serine specific, 0.25 mM) and E-64 (cysteine specific, 10 μM). Inhibitor/agonist and or HDM added at the same time. Each dot represents one well and data shown for mean ± SEM. One-way ANOVA for (C, D, E); * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Symbol on top of multiple brackets represents all brackets. Agonist, PAR1 agonist vorapaxar; ePNEC, epithelial-derived pulmonary neuroendocrine cells; Inh., PAR1 inhibitor; iPNEC, iPSC-derived pulmonary neuroendocrine cells; HDM, House dust mite; PAR1, Protease activated receptor 1; SYP, Synaptophysin.

Article Snippet: Cells were grown using the bronchial epithelial cell growth kit from ATCC (PCS300- 040).

Techniques: Single-cell Transcriptomics, Immunohistochemical staining, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Derivative Assay

Journal: Nature

Article Title: Virus-induced senescence is a driver and therapeutic target in COVID-19.

doi: 10.1038/s41586-021-03995-1

Figure Lengend Snippet: Fig. 5 | Senolytic targeting mitigates disease features of COVID-19 in animal models and patients. a, H3K9me3 IHA in nasopharyngeal (top) and lung (middle) specimens, and lipofuscin-based detection (bottom) of senescent cells in the lungs of golden hamsters infected with SARS-CoV-2 and treated with navitoclax or solvent-only (n = 5 animals each), or mock-infected and untreated (n = 3 animals) at day 7 after infection. b, H3K9me3 IHA and lipofuscin staining in nasopharyngeal sections of Roborovski dwarf hamsters infected with SARS-CoV-2 or mock at day 4 after infection and treatment as indicated. c, Lung tissue of dwarf hamsters as in b on day 4 after infection stained with haematoxylin and eosin. Uninfected healthy hamsters (mock) are shown for comparison ((xiv)–(xvii)). (i), (v) and (x) are overviews of lung sections. Signs of COVID-19-like pneumonia and haemorrhage are indicated: hyperplasia of the bronchial epithelium (double-headed arrows), haemorrhage (asterisks) and necrosis (black arrowheads) of alveolar epithelial cells with neutrophil infiltration (white arrowheads), and endothelialitis (black arrows).

Article Snippet: Primary human nasal or bronchial epithelial cells (HNEpC and HBEpC) were purchased from PromoCell.

Techniques: Infection, Solvent, Staining, Comparison

Journal: Nature Communications

Article Title: PMI-controlled mannose metabolism and glycosylation determines tissue tolerance and virus fitness

doi: 10.1038/s41467-024-46415-4

Figure Lengend Snippet: a Growth curves of A549-wildtype (WT) and knockout ( PMI -/- ) cells supplemented with 0, 5, or 25 mM of mannose ( n = 4 biological repeats). b Bioenergetic profiles of H1N1-infected WT and PMI -/- cells supplemented with or without mannose (25 mM) treatment. Cells were pre-treated with mannose overnight before virus infection for another 12 h. Glycolytic rate of the cells were analyzed using a Seahorse XF analyzer by measuring the glycoPER kit ( n = 5 biological repeats). c Mitochondrial respiration of the cells was analyzed using a Seahorse XF analyzer by measuring the oxygen consumption rates (OCR, n = 5 biological repeats). d Either PMI overexpression (O/E) or supplement of PMI metabolite Fructose-6-phosphate (F6P) antagonize the suppression of low mitochondrial membrane potential (MMP) by mannose. A549 cells were pre-treated with the indicated treatment for overnight before H1N1 infection (MOI = 2). After 12 h, cells were subject to MMP analysis after JC-1 staining ( n = 3 biological repeats). e Dual PMI depletion and mannose treatment is unfavorable whereas supplement of F6P reverses the mitochondrial damage. A549-WT and PMI –/– cells were pre-treated with the indicated treatment, followed by H1N1 infection and MMP measurement after JC-1 staining ( n = 3 biological repeats). f MMP assay by TMRE Staining. hBTEC (left panel) and A549 cells (right panel) were pre-incubated with the indicated treatment for 12 h before H1N1 virus infection (MOI = 0.2). Cells were subject to TMRE (200 nM) staining for 20 min, and DAPI staining for normalization. The fluorescence intensities were detected by a plate reader, whereas the images were captured by GE IN Cell Analyzer 6500HS ( n = 3 biological repeats). g Knockout of PMI impair the modulatory activity of mannose against IL-1β production. The experiments were performed in WT and PMI –/– cells transfected with PMI plasmid and/or Poly(I:C) as indicated ( n = 3 biological repeats). All the results are shown as mean ± SD. One-way ANOVA with Dunnett’s post hoc test was used for ( a , d , e ). Unpaired and two-sided student’s T test was used for ( f , g ). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and n.s. indicates non-significant.

Article Snippet: The Bronchial/Tracheal Epithelial Cells (hBTEc) were obtained from the ATCC company (ATCC PCS-300-010) and were cultured with ATCC complete growth media, consisting of ATCC Airway Epithelial Cell Basal Medium (ATCC PCS-300-030) supplemented with Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040).

Techniques: Knock-Out, Infection, Virus, Over Expression, Membrane, Staining, Mmp Assay, Incubation, Fluorescence, Activity Assay, Transfection, Plasmid Preparation

Journal: Cell reports

Article Title: Interferon (IFN)-γ promotes monocyte-mediated lung injury during influenza infection

doi: 10.1016/j.celrep.2022.110456

Figure Lengend Snippet: Key Resources Table

Article Snippet: Primary Bronchial/Tracheal Epithelial Cells; Normal, Human , ATCC , Cat#PCS-300-01.

Techniques: Virus, Recombinant, cDNA Synthesis, SYBR Green Assay, Protein Extraction, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Software, Microscopy, Flow Cytometry, Fluorescence

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Electronic Nicotine Delivery System Aerosol-induced Cell Death and Dysfunction in Macrophages and Lung Epithelial Cells

doi: 10.1165/rcmb.2019-0200oc

Figure Lengend Snippet: Figure 1: ENDS aerosol exposure causes increased cell death in human lung epithelial cells The BEAS-2B cell line was exposed to ENDS aerosol with (PG/VG N+) and without nicotine (PG/VG N-), 100μM Nicotine (Nicotine), HEPA filtered air (Air), and room temperature (Untreated) for 4 min. Cell death was analyzed 24h after treatment by flow cytometry using Caspase-3/7 stain and SYTOXTM permeability dye. Representative plots are shown in (A) and quantification show in (B-E). Necrotic cells are defined as SYTOXTM +, Caspase-3/7- (i) (B). Secondary necrotic cells are dual positive (ii) (C). Apoptotic cells are Caspase-3/7+, SYTOXTM- (iii) (D). All cells with activated Caspase-3/7 (E). Experiments were performed 3 times with an n > 3 per group. *=p<0.05 compared to untreated, #=p<0.05 compared to air, &=p<0.05 compared to PG/VG N-, %=p<0.05 compared to Nicotine. Data were analyzed using ANOVA followed by Tukey’s multiple comparisons test. Mean values are shown with SEM.

Article Snippet: Primary human bronchial epithelial cells (ATCC® PCS-300-010TM) were cultured on 24-well transwell inserts and grown in PneumaCultTM Medium system (Stemcell Technologies Inc.).

Techniques: Aerosol, Flow Cytometry, Staining, Permeability

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Electronic Nicotine Delivery System Aerosol-induced Cell Death and Dysfunction in Macrophages and Lung Epithelial Cells

doi: 10.1165/rcmb.2019-0200oc

Figure Lengend Snippet: Figure 2: ENDS aerosol exposure causes increased cell death in mouse lung epithelial cells The MLE12 cell line was exposed to ENDS aerosol with (PG/VG N+) and without nicotine (PG/VG N-), 100μM Nicotine (Nicotine), HEPA filtered air (Air), and room temperature (Untreated) for 4 min. Cell death was analyzed 24h after treatment by flow cytometry using Caspase-3/7 stain and SYTOXTM permeability dye. Representative plots are shown in (A) and quantification show in (B-E). Necrotic cells are defined as SYTOXTM +, Caspase-3/7- (i) (B). Secondary necrotic cells are dual positive (ii) (C). Apoptotic cells are Caspase-3/7+, SYTOXTM- (iii) (D). All cells with activated Caspase-3/7 (E). Experiments were performed 3 times with an n > 3 per group. *=p<0.05 compared to untreated, #=p<0.05 compared to air, &=p<0.05 compared to PG/VG N-, %=p<0.05 compared to Nicotine. Data were analyzed using ANOVA followed by Tukey’s multiple comparisons test. Mean values are shown with SEM.

Article Snippet: Primary human bronchial epithelial cells (ATCC® PCS-300-010TM) were cultured on 24-well transwell inserts and grown in PneumaCultTM Medium system (Stemcell Technologies Inc.).

Techniques: Aerosol, Flow Cytometry, Staining, Permeability

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Electronic Nicotine Delivery System Aerosol-induced Cell Death and Dysfunction in Macrophages and Lung Epithelial Cells

doi: 10.1165/rcmb.2019-0200oc

Figure Lengend Snippet: Figure 3: One week of ENDS aerosol exposure causes decreased trans-epithelial resistance, dissociation of cells, and cell death Primary human bronchial epithelial cells were differentiated on transwell inserts into a pseudostratified epithelial layer. These cells were exposed to ENDS aerosol with (PG/VG N+) and without nicotine (PG/VG N- ), HEPA filtered air (Air), 100uM nicotine (Nicotine), and room temperature (Untreated) for 4 min. Trans- epithelial resistance was measured using a WPI EVOM TEER meter and STX3 electrode. Measurements were taken in triplicate and averaged. Background readings of empty wells as well as baseline measurements of each sample were used to calculate the change in resistance over the course of the treatment. Dissociated and dead cells were detected using trypan blue exclusion and counting on a hemocytometer. Treatments occurred once per day for 7 days. Experiments were performed 2 times with an n > 3 per group. *=p<0.05 compared to untreated, #=p<0.05 compared to air, %=p<0.05 compared to Nicotine. Data were analyzed using ANOVA followed by Tukey’s multiple comparisons test. Mean values are shown with SEM.

Article Snippet: Primary human bronchial epithelial cells (ATCC® PCS-300-010TM) were cultured on 24-well transwell inserts and grown in PneumaCultTM Medium system (Stemcell Technologies Inc.).

Techniques: Aerosol

Journal: Annals of Medicine

Article Title: Hsa_circ_0042823 accelerates cancer progression via miR-877-5p/FOXM1 axis in laryngeal squamous cell carcinoma

doi: 10.1080/07853890.2021.1934725

Figure Lengend Snippet: Hsa_circ_0042823 was up-regulated in AMC-HN-8 and TU686 cells. (A) The expression of hsa_circ_0042823 in HBECs, AMC-HN-8 and TU686 cells was assessed through qRT-PCR. (B and C) The qRT-PCR was performed to detect hsa_circ_00428233 expression in the AMC-HN-8 and TU686 cells after hsa_circ_0042823 overexpression or knockdown. ** p < .01 compared with HBECs; ## p < .01 compared with Vector; $$ p < .01 compared with Si-NC.

Article Snippet: Human bronchial epithelial cells (HBECs) were purchased from ATCC (Cat: PCS-300-013, Manassas, VA).

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Knockdown, Plasmid Preparation

Journal: Pharmaceuticals

Article Title: Corylin Ameliorates LPS-Induced Acute Lung Injury via Suppressing the MAPKs and IL-6/STAT3 Signaling Pathways

doi: 10.3390/ph14101046

Figure Lengend Snippet: Anti-inflammatory effect of corylin on LPS-induced ALI. The experimental results demonstrated that corylin attenuated the overproduction of IL-6 in LPS-activated human bronchial epithelial cells. In intratracheal LPS-induced ALI mice, corylin attenuated tissue damages, suppressed inflammatory cell infiltration, and decreased secretion of IL-6 and TNF-α in the BALF and serum; moreover, it further inhibited the expression of phosphorylation of mitogen-activated protein kinases (MAPKs), including the expression of p-JNK/JNK, p-ERK/ERK, p-p38/p38, and repressed the activation of signal transducer and activator of transcription 3 (STAT3) in lung. Taken together, our results are the first to demonstrate the anti-inflammatory effects of corylin on LPS-induced ALI and suggest corylin has significant potential as a novel therapeutic agent for ALI.

Article Snippet: A human bronchial epithelial cell line, HBEC3-KT cells, was purchased from American Type Culture Collection (No. CRL-4051, Manassas, VA, USA).

Techniques: Expressing, Phospho-proteomics, Activation Assay

Journal: Cancers

Article Title: Control of Mitochondrial Electron Transport Chain Flux and Apoptosis by Retinoic Acid: Raman Imaging In Vitro Human Bronchial and Lung Cancerous Cells.

doi: 10.3390/cancers15184535

Figure Lengend Snippet: Figure 2. Raman imaging of a typical human bronchial epithelial lung cell (BEpC) without (control) and incubated with retinoic acid (50 µM) for 48 h. Microscopy images (A,E), Raman images with the respective Raman spectra (B,D,F,H) of a nucleus (red), lipid droplets (orange), endoplasmic reticulum (blue), cytoplasm (green), mitochondria (magenta), cell border (light grey). Fluorescence images (C,G). Resolution of Raman images 1 µm, integration time 0.3 s, 10 mW at 532 nm and resolution of fluorescence images 1 µm, integration time 0.01 s (Nucleus—blue (Hoechst 33342), ER/lipid droplets—red (Oil Red O)).

Article Snippet: BEpC cells were grown in Bronchial Epithelial Cell Medium (no. P60151, Innoprot) and maintained at 37 ◦C in a humidified atmosphere containing 5% CO2 according the Innoprot’s protocol.

Techniques: Imaging, Control, Incubation, Microscopy, Fluorescence