a549 nf κb luc cells  (Panomics Inc)

Journal: Respiratory Research

Article Title: Aggregatibacter is inversely associated with inflammatory mediators in sputa of patients with chronic airway diseases and reduces inflammation in vitro

doi: 10.1186/s12931-024-02983-z

Figure Lengend Snippet: Determination of the minimal effective MOI (mMOI) of a ) A. actinomycetemcomitans and b ) A. aphrophilus based on NF-κB pathway activation and determination of the mMOI of c ) A. actinomycetemcomitans and d ) A. aphrophilus based on IL-8 production in 3-D lung epithelial cells. On the vertical axis % NF-kB pathway activation / IL-8 production compared to LPS-stimulated cells is shown, on the horizontal axis the tested MOI is depicted. The mMOI was defined as the lowest MOI with anti-inflammatory activity (i.e. < 50% NF-kB pathway activation / IL-8 production compared to LPS-stimulated cells as visualized by a dotted line, p < 0,05). e ) Western blot analysis of proteins (i.e. IκBα, p65 and phosphorylated (p)-IκBα) produced by 3-D A549 cells stimulated with LPS in the presence or absence of A. actinomycetemcomitans (targeted MOI 50:1) for 4 h (representative replicate is shown, image contains cropped blot – the full original blot is shown in Supplementary file_Western blot image). f ) Band intensity (normalised to β-actin) of Western blot at 4 h of 3-D A549 cells stimulated with LPS with/without A. actinomycetemcomitans . Results are expressed as a percentage of the positive control (i.e. LPS). NC: negative control (untreated 3-D A549 cells in serum-free GTSF-2 medium), A.ac. A. actinomycetemcomitans . Data represent mean ± SEM, * p < 0.05, n ≥ 3

Article Snippet: A previously developed organotypic 3-D lung cell culture model of the A549 alveolar epithelial cell line (ATCC CCL185) or the NF-κB–luciferase-transfected A549 recombinant stable cell line (BPS Bioscience, San Diego, CA, US) was used that exhibits in vivo-like phenotypic and functional properties of alveolar epithelial cells, including barrier function (localized expression of tight junctional markers), apical and basolateral polarity, and responds to infection in ways that are relevant to the infection process in vivo, including cytokine secretion [ , – ].

Techniques: Activation Assay, Activity Assay, Western Blot, Produced, Positive Control, Negative Control

Journal: Mucosal immunology

Article Title: Noxa/HSP27 Complex Delays Degradation of Ubiquitylated IkBα in Airway Epithelial Cells to Reduce Pulmonary Inflammation

doi: 10.1038/mi.2017.117

Figure Lengend Snippet: AALEB cells were infected with Ad-Noxa or Ad-GFP (80 MOI) and were treated or left untreated with 20 ng/ml of TNFα for 24 h. Secreted IL-8 was quantified by Luminex assay (n= 3 experiments with 3 wells in each experiment). (B) Luciferase activity in A549-Luc cells when either infected with 100, 200 or 300 MOI Ad-Noxa or 300 MOI Ad-LacZ and stimulated with TNFα for 30 min, or (C) when the cells were stably transfected with shNoxa or shCtrl and treated with IFN-γ; reduced expression of Noxa mRNA and protein by shNoxa was verified by qRT-PCR and Western blotting. (D) AALEB cells treated with IFN-y (50 ng/ml) or vehicle and 24 h later with TNFα for 30 min. Cells immunostained with antibodies to NF-κB and Noxa and counterstained with Hoechst 33342. (E) Immunostaining with antibodies to NF-κB and Noxa of AALEB cells infected with Ad-Noxa or Ad-LacZ treated with TNFα for 30 min; percentage of cells with nuclear NF-κB. Error bar indicates ± SEM (n=3 independent experiments with 3 wells in each experiment). * P < 0.05, ** P < 0.01, *** P <0.001. (F) Cytosolic and nuclear extracts from noxa +/+ and noxa -/- MAECs treated with IFN-γ and TNFα and analyzed by Western blotting. (G) AALEB cells infected with Ad-Noxa and Ad-GFP and treated with cyclohexamide. Extracts probed for NF-κB, IκB, Noxa, and GAPDH. (H+I) AALEB cells infected with Ad-GFP or Ad-Noxa, treated with 10 μM MG-132 and 30 min later with vehicle or 10 ng/ml TNFα; cells harvested 10 min later and extracts immunoprecipitated with anti-IκBα antibodies. (H) The levels of p-IκBα and Noxa levels were determined with the respective controls in input and immunoprecipitates. (I) Levels of p-IκBα and Noxa levels in the input and IκBα and K48 chain-linked ubiquitin of IκBα were determined in the immunoprecipitates. Western blots are representative of 3 different experiments.

Article Snippet: A549 /NF-κB-luc cell line (Panomics Corp.) was used to monitor the activity of NF-κB transcription factor in a cell based assay (R/D Biosystems, Minneapolis, MN).

Techniques: Infection, Luminex, Luciferase, Activity Assay, Stable Transfection, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Immunostaining, Immunoprecipitation, Ubiquitin Proteomics

Journal: Mucosal immunology

Article Title: Noxa/HSP27 Complex Delays Degradation of Ubiquitylated IkBα in Airway Epithelial Cells to Reduce Pulmonary Inflammation

doi: 10.1038/mi.2017.117

Figure Lengend Snippet: (A) Luciferase activity in A549-Luc cells treated with vehicle or peptides A, B, C, and D at 50 nM concentration. (B) Nuclear localization of NF-κB (p65) following treatment of noxa -/- MAECs with 0 or 10 ng/ml TNFα in the presence of TAT-Ctrl, and peptides A or D. (C + D) C57Bl/6 mice exposed to 50 μg house dust mite for 5 consecutive days and instilled with 10 μM each of TAT-Ctrl or peptide A on days 6 and 7. On day 8, the numbers of total inflammatory cells (C) and eosinophils (D) were assessed in the BAL fluid. Error bar indicates ± SEM (in 2 separate experiments with minimum of 4 mice/group each). * P < 0.05. (E) AALEB cells were treated with TAT-Ctrl or peptide A and 24 h later treated with TNFα for 10 min. Immunoprecipitates with anti-IκBα antibodies were analyzed for levels of IκBα and K48 chain linked ubiquitin of IκBα and IκBα and β-actin in the input. Western blots are representative of 3 different experiments.

Article Snippet: A549 /NF-κB-luc cell line (Panomics Corp.) was used to monitor the activity of NF-κB transcription factor in a cell based assay (R/D Biosystems, Minneapolis, MN).

Techniques: Luciferase, Activity Assay, Concentration Assay, Ubiquitin Proteomics, Western Blot