complete bronchial epithelial cell growth medium  (Lonza)

Journal: Frontiers in Immunology

Article Title: CYP1B1 knockout enhanced IFN-γ production is required but not sufficient for protection of cigarette smoke-exposed mice against lethal influenza virus infection

doi: 10.3389/fimmu.2025.1600025

Figure Lengend Snippet: CYP1B1 is induced by IAV and CSE in BciNS1 cells, and TMS protects airway epithelial cells from cell death induced by IAV infection. BciNS1 cells were incubated with 0% (Mock) and 5% CSE for 24h. For IAV infection, the cells were exposed to IAV PR8 at an MOI of 0.1 for 24h. (A) CYP1B1 mRNA levels were assessed by qRT-PCR and normalized to β-actin. (B) CYP1B1 protein expression in BciNS1 cells was determined by western blot. Data are expressed as means ± SD. * denotes a significant difference compared to the mock group ( P < 0.05 by one-way ANOVA with Tukey test for multiple comparisons, n = 3.). (C) LDH activity in the supernatant and cell extract was measured using an LDH Cytotoxicity Assay Kit (BioVision Research Products). Cytotoxicity is expressed as the percentage of supernatants released LDH to total (supernatant + cell) LDH. * denotes a significant difference between the two groups ( P < 0.05 by one-way ANOVA with Tukey test for multiple comparisons, n = 3).

Article Snippet: Cells were resuspended to 5 × 10 5 cells/ml in complete Bronchial Epithelial Cell Growth Medium (BEGM; Lonza Group Ltd.), were seeded into collagen-coated tissue culture plates (Bio-Coat, BD Biosciences) at a density of 1 × 10 5 cells/cm 2 , and were propagated in an incubator at 37°C in 5% CO 2 .

Techniques: Infection, Incubation, Quantitative RT-PCR, Expressing, Western Blot, Activity Assay, LDH Cytotoxicity Assay

Journal: eBioMedicine

Article Title: Cross-sectional and longitudinal genotype to phenotype surveillance of SARS-CoV-2 variants over the first four years of the COVID-19 pandemic

doi: 10.1016/j.ebiom.2024.105415

Figure Lengend Snippet: Preferential usage of TMPRSS2 cleavage resistant ACE2 over time and increased replication of JN.1 versus XBB.1.5 in primary upper respiratory epithelial cultures. a. The structure of the ACE2 dimer is presented (blue) based on the published structure, with the TMPRSS2 cleavage site (pink) and the cleavage sequence (boxed in red). TMPRSS2 (green) cleavage events within ACE2 and SARS-CoV-2 Spike glycoprotein (highlighted by arrows). b. The structure of the ACE2 dimer is presented as a dimer of heterodimers complexed with solute carrier SLC6A19 (dark blue) based on the published structure. ACE2 complexes with either solute carriers SLC6A20 and SLC6A19 in and around the TMPRSS2 cleavage site, which is predicted to structurally occlude TMPRSS2 from accessing this site for cleavage. To control for lack of TMPRSS2 cleavage, an ACE2 cleavage resistant mutant (NC-ACE2) was generated, as previously described. c. Both cleavage sensitive (WT ACE2) and cleavage resistant (NC-ACE2) were expressed alongside TMPRSS2 in the VeroE6 cell line. Both Ancestral Clade A.2.2 and JN.1 were titrated in both cell lines, with cytopathic effects presented after 2 days of culture. The top two rows of images demonstrate viral dilutions of Clade A.2.2 whilst the bottom two rows of images reflect JN.1 viral dilutions. d & e . Representative titration curves using cells expressing d. wild type ACE2 and e . TMPRSS2 cleavage resistant ACE2 and TMPRSS2. For d and e . viral replication is determined through dose dependent loss of live cell nuclei 3 days post infection. Standard deviations represent 8 technical replicates. f. Augmentation versus attenuation are presented for the major circulating variants throughout the pandemic (ancestral Clade A.2.2, Alpha, Beta, Delta, Omicron BA.1, BA.2, BA.5, XBB.1, CH.1.1, XBF, XBB.1.5, and JN.1). Titers are calulated here in the parental VeroE6-TMPRSS2 cell line alongside the WT ACE2 and NC-ACE2 cell lines shown in C. The red line signifies the point where the virus is replicating in the cell line at a level equivalent to its parent cell line VeroE6-TMPRSS2. ∗ = P < 0.05 for decrease in attenuation in the NC-ACE2 versus WT ACE2 setting. ∗∗∗∗ = P < 0.0005 for significant rescue in the NC-ACE2 versus WT ACE2 setting g. Augmentation and attenutation as outlined in f. presented with respect to time with representative lineages circulating in 2023. Decling use of cleavage sensitive ACE2 is presented (red line). h. Viral loads derived from primary ALI cultures. Clinical isolates XBB.1.5 and JN.1 were expanded and then equal RNA copies/ml was used to inoculate primary ALI cultures. Supernatants were harvested and RNA loads measured. Shown are the mean ± s.d. from n = 3 experiments ∗∗ = P < 0.005.

Article Snippet: All participants underwent fibre-optic bronchoscopy in accordance with standard guideline. pBEC cultures were grown and differentiated until confluent in complete Bronchial Epithelial Cell Growth Basal Medium (Lonza, CC-3171) before use for air–liquid interface (ALI) experiments.

Techniques: Sequencing, Control, Mutagenesis, Generated, Titration, Expressing, Infection, Virus, Derivative Assay

Journal: Inflammation and Regeneration

Article Title: Tenascin-C modulates alveolarization in bronchopulmonary dysplasia

doi: 10.1186/s41232-024-00330-9

Figure Lengend Snippet: HIF-1α directly induces soluble TN-C expression in epithelial cells. A - B Soluble TN-C (sTN-C) was measured via Western blotting, and Ponceau S was used as a loading control to assess the total protein concentration in the cell culture supernatant. C HIF-1α protein expression in MLE-12 cells stimulated with 85% oxygen for 12 h. D - E TN-C protein expression in MLE-12 cells stimulated with the HIF-1α agonist DMOG under normoxia ( D ) or the HIF-1α inhibitor 2-MeOE2 under 85% hyperoxia ( E ) for 12 h was measured via Western blotting. The density quantification of TN-C or HIF-1α was expressed as a ratio relative to β-actin. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: BEAS-2B cells (purchased from Procell Life Science & Technology Co. Ltd.), a kind of human bronchial epithelium, were cultured in complete bronchial epithelial cell growth medium (Lonza, CC-3170 and CC-4175).

Techniques: Expressing, Western Blot, Control, Protein Concentration, Cell Culture

Journal: Inflammation and Regeneration

Article Title: Tenascin-C modulates alveolarization in bronchopulmonary dysplasia

doi: 10.1186/s41232-024-00330-9

Figure Lengend Snippet: Low-concentration sTN-C supplementation enhanced the proliferation and migration of epithelial cells. A The overexpression of sTN-C in the supernatant of transfected 293 T cells was verified by Western blotting. B CCK-8 assay for determining the viability of MLE-12 cells incubated for 24 h under normoxia after low-dose sTN-C supplementation (2 ng/mL). C , E . CCK-8 assay was used to assess the viability of MLE-12 cells or BEAS-2B cells incubated for 24 h under 85% hyperoxia following low-dose supplementation with sTN-C (2 ng/mL) and TN-C neutralizing antibodies. D , F EdU assays were used to assess the proliferation of MLE-12 and BEAS-2B cells incubated for 24 h under 85% hyperoxia following low-dose supplementation with sTN-C (2 ng/mL) and TN-C neutralizing antibodies (2 ng/mL). Scale bars = 50 μm. G - H A live cell imaging system was used to observe the migration ability of alveolar epithelial cells at the scratch edge after a 90-min incubation period under normoxia following the addition of low-dose sTN-C (2 ng/mL). Scale bars = 50 μm. The average migration speed of individual cells was analyzed using ImageJ. All experiments were repeated at least 3 times. *** P < 0.001, **** P < 0.0001

Article Snippet: BEAS-2B cells (purchased from Procell Life Science & Technology Co. Ltd.), a kind of human bronchial epithelium, were cultured in complete bronchial epithelial cell growth medium (Lonza, CC-3170 and CC-4175).

Techniques: Concentration Assay, Migration, Over Expression, Transfection, Western Blot, CCK-8 Assay, Incubation, Live Cell Imaging

Journal: Inflammation and Regeneration

Article Title: Tenascin-C modulates alveolarization in bronchopulmonary dysplasia

doi: 10.1186/s41232-024-00330-9

Figure Lengend Snippet: The potential role of ICAM-1 in the proliferation of alveolar epithelial cells stimulated by low-dose sTN-C. 2 ng/mL sTN-C (a blank vector-transfected supernatant as a control) was used to activate MLE-12 cells in 24-well plates for 24 h. Total RNA was extracted using TRIzol, after which RNA sequencing was carried out. A Process diagram for transcriptome sequencing. B Bubble diagram of KEGG pathway enrichment. The AGE-RAGE signaling pathway was considerably increased in low-dose sTN-C-treated alveolar epithelial cells. C Volcano plot showing DEGs in sTN-C-treated alveolar epithelial cells. The upregulated genes are shown in red, and the downregulated genes are shown in green with a fold change > 1.0 and P < 0.05. ICAM-1 is marked with a special color. D The increase in ICAM-1 RNA expression was verified by qPCR. E ICAM-1 RNA expression in MLE-12 cells following 24 h of stimulation with 85% oxygen was measured by qPCR. F ICAM-1 RNA expression levels in the lung tissue of BPD model mice at P7 or P14 were measured via qPCR. G ICAM-1 protein expression in pulmonary tissues at P14 was measured by Western blotting. H In a model of alveolar epithelial cells stimulated with low-dose sTN-C, anti-ICAM-1 monoclonal antibodies (2 μg/mL) and isotype mouse IgG control antibodies (2 μg/mL) were added. The proliferation of MLE-12 cells was assessed using a CCK-8 assay

Article Snippet: BEAS-2B cells (purchased from Procell Life Science & Technology Co. Ltd.), a kind of human bronchial epithelium, were cultured in complete bronchial epithelial cell growth medium (Lonza, CC-3170 and CC-4175).

Techniques: Plasmid Preparation, Transfection, Control, RNA Sequencing, Sequencing, RNA Expression, Expressing, Western Blot, Bioprocessing, CCK-8 Assay

Journal: Inflammation and Regeneration

Article Title: Tenascin-C modulates alveolarization in bronchopulmonary dysplasia

doi: 10.1186/s41232-024-00330-9

Figure Lengend Snippet: Excess TN-C slowed alveolar formation. A CCK-8 assay for determining the viability of MLE-12 cells incubated for 24 h under normoxia after low-dose sTN-C supplementation (8 ng/mL). B - C The migration ability of alveolar epithelial cells was measured by the scratch method. In a mouse model of BPD, newborn mice at P2, P4, P6, P8, and P10 received continual intraperitoneal injections of isotype rat IgG antibodies or TN-C neutralizing antibodies at a concentration of 1 μg/body. D - F Lung morphometry was analyzed via H&E staining of tissue samples from surviving pups at P14. Scale bar = 100 μm. G RAGE and SP-C protein expression in lung tissues. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: BEAS-2B cells (purchased from Procell Life Science & Technology Co. Ltd.), a kind of human bronchial epithelium, were cultured in complete bronchial epithelial cell growth medium (Lonza, CC-3170 and CC-4175).

Techniques: CCK-8 Assay, Incubation, Migration, Concentration Assay, Staining, Expressing