Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: The release of neutrophil extracellular traps (NETs) by neutrophils promotes migration and invasion of NSCLC. (A) The morphology of neutrophils isolated from healthy donors’ blood was observed by Giemsa staining (magnification, 1000×). (B) The neutrophil viability was assessed by trypan blue dye exclusion assays (magnification, 100×). (C) Representative images and quantification of NETs formation of neutrophils from healthy donors (HD) and NSCLC patients. MPO (red), cit-H3 (green), and DAPI (blue), respectively (magnification, 50×; scale bar, 200μm). (D) Representative images of NETs formation in NSCLC patients’ normal lung tissues and tumor tissues that were detected by con-focal microscopy. cit-H3 (red), Ly6g (green), and DAPI (blue), respectively (magnification, 200×; scale bar, 100µm and magnification, 400×; scale bar, 50µm). (E) Representative images of PMA-induced NETs formation of neutrophils from HD stained with MPO and cit-H3 were detected by immunofluorescence microscope; MPO (red), cit-H3 (green), and DAPI (blue), respectively (magnification, 50×; scale bar, 200μm). Transwell invasion (F) and wound healing assays (G) were performed to identify the effects of NETs on A549 and SK-MES-1 cells invasion (magnification, 100×) and migration (magnification, 50×). (H) Western blot analyzing the expressions levels of EMT markers protein (N-cadherin, E-cadherin, and Vimentin) in A549 and SK-MES-1 cells treated with NETs. ( * P < 0.05, ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Migration, Isolation, Staining, Microscopy, Immunofluorescence, Western Blot

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: MIR503HG is downregulated in NSCLC cells with NETs stimulation and is associated with poor survival of NSCLC. (A) Volcano plot illustrating the differentially expressed lncRNAs in A549 cells treated with or without NETs for 12 h (|log 2 fold change (FC)| > 2, P -value < 0.01). (B) Heat map showing the top 30 differentially down-regulated lncRNAs in A549 cells after treatment with NETs for 12 h. Red means up-regulated, blue means down-regulated, separately. (C) Relative expression of MIR503HG in A549 and SK-MES-1 cells with or without NETs stimulation. (D) MIR503HG is expressed at a lower level in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tumor compared to corresponding adjacent normal lung tissues according to the TCGA database. (E) The expression of MIR503HG in 50 paired NSCLC tumors and normal tissues were quantified by qRT-PCR. (F) Kaplan-Meier analysis of metastasis risk of 50 NSCLC patients divided into two groups based on a middle cutoff of MIR503HG expression. (G) MIR503HG is mainly located in the nuclear of NSCLC cells. U6 snRNA (nuclear reserved) and GAPDH mRNAs (exposed to cytoplasm) were used as controls. Data are mean ± SD (n=3). ( ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: Overexpression of MIR503HG substantially reversed the metastasis-promoting effect of NETs on NSCLC in vitro and vivo. (A) Expression of MIR503HG was successfully up-regulated in A549 and SK-MES-1 cells. (B, C) Wound healing (magnification, 50×) and invasion assays (magnification, 100×) of NSCLC cells that stable transfection of MIR503HG vector versus control vector both treat with NETs 12 h. (D) Western blot analysis of the expression of EMT (N-cadherin, E-cadherin, Vimentin) in control and MIR503HG overexpressing A549 and SK-MES-1 cells after NETs treated 12 h. (E) Schematic diagram showing the experimental design of the effect of MIR503HG on NETs-induced metastasis. Representative images of the gross lung (F) and H&E staining (G) of metastatic lung nodules in mice specimens. (H) Quantification of the number, volume, and maximum size of metastatic lung nodules. Data are mean ± SD (n=5 nude mice in each group). (magnification, 100×; scale bar, 200 μm, magnification, 200×; scale bar, 50 μm). (I) Immunohistochemistry (IHC) detection of N-cadherin, E-cadherin, and Vimentin revealed EMT formation in the NETs-induced lung metastasis model (magnification, 100× and 400×). ( ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Over Expression, In Vitro, Expressing, Stable Transfection, Plasmid Preparation, Control, Western Blot, Staining, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: NETs promote migration and invasion of NSCLC by activating the NLRP3 inflammasome. (A) Up-regulated NSCLC-related pathways in response to NETs stimulated revealed by KEGG enrichment. (B) The mRNA expression of NLRP3, Caspase1, IL-1β and IL-18 in A549 cells was analyzed by qRT-PCR after NETs stimulation at different times. (C) Western blotting was used to analyze the expression of NLRP3 and Caspase1 in A549 and SK-MES-1 cells after NETs stimulation for different periods. (D) Immunofluorescence was used to observe the expression of ROS in A549 and SK-MES-1 cells treated with NETs for 12 h (magnification, 100×). The expression of NLRP3, Caspase1, IL-1β and IL-18 were detected by qRT-PCR (E) and Western blot (F) in A549 and SK-MES-1 cells after treating with NETs and NLRP3 inflammasome inhibitor MCC950, respectively. (G–I) Estimate the effect of NETs on the level of N-cadherin, E-cadherin, and Vimentin (Western blot) (G) in A549 and SK-MES-1 cells and the capacity of invasion (transwell invasion assays; magnification, 100×) (H) , migration (wound healing assays; magnification, 50×) (I) when inhibiting the expression of the NLRP3 inflammasome by MCC950. ( * P < 0.05, ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Migration, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: NLRP3 inflammasome mediated the effect of MIR503HG to inhibit NETs-triggered metastasis of NSCLC. (A, B) The protein and mRNA expression of NLRP3 and Caspase1 in A549 and SK-MES-1 cells with MIR503HG overexpression were analyzed by Western blot (A) and qRT-PCR (B) after NETs were stimulated. (C) A negative relationship between MIR503HG and NLRP3 in NETs-induced NSCLC cells is presented by correlation analysis. (D) Overexpression of NLRP3 attenuated the effect of MIR503HG in inhibiting NETs-triggered EMT in NSCLC cells by Western blot. (E, F) Overexpression of NLRP3 effectively reverses the effect of MIR503HG in inhibiting NETs-triggered promotion of NSCLC cells metastasis using transwell assay (magnification, 100×) (E) and wound healing assays (magnification, 50×) (F) . ( * P < 0.05, ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Expressing, Over Expression, Western Blot, Quantitative RT-PCR, Transwell Assay

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: NLRP3 inflammasome induced by NETs promotes NSCLC progression is associated with the activation of NF-κB. (A) The protein expression level of p-p50, p50, p-p65, p65 in NSCLC cells treated with NETs was detected by Western blotting. (B) Nuclear translocation of NF-κB in A549 and SK-MES-1 cells treated with NETs or combined with DNase I were detected by con-focal microscopy (magnification, 3000×; scale bar, 5μm). Immunofluorescence assays (C) and Western blot (D) were used to detect the effect of NETs on NLRP3 inflammasome in A549 and SK-MES-1 cells after p50 knockdown (magnification, 200×; scale bar, 50 μm). (E) Downregulation of p50 attenuated the effect on promoting EMT of NETs in NSCLC cells by Western blot. (F, G) Downregulated p50 reverses NETs-induced promotion of NSCLC cells metastasis using transwell assay (magnification, 100×) (E) and wound healing assays (magnification, 50×) (F) .

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Activation Assay, Expressing, Western Blot, Translocation Assay, Microscopy, Immunofluorescence, Knockdown, Transwell Assay

Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps (NETs) Promote Non-Small Cell Lung Cancer Metastasis by Suppressing lncRNA MIR503HG to Activate the NF-κB/NLRP3 Inflammasome Pathway

doi: 10.3389/fimmu.2022.867516

Figure Lengend Snippet: MIR503HG inhibits NETs-triggered NSCLC cells metastasis capacity and NLRP3 inflammasome activation dependently on NF-κB. (A) Western blotting was used to detect the changes of p-p50, p50, p-p65 and p65 in MIR503HG overexpression A549 and SK-MES-1 cells after NETs treatment. (B) qRT-PCR and (C) Western blotting analyses of up-regulating p50 in A549 and SK-MES-1 cells. (D) Analysis of the NLRP3 and Caspase1 protein levels in MIR503HG-overexpressed NSCLC cells transfected with p50-pcDNA3.1 and pcDNA3.1 vector by western blot with NETs stimulated. Transwell invasion (magnification, 100×) (E) and wound healing assays (magnification, 50×) (F) were performed to identify the effects of NETs on MIR503HG-overexpressed NSCLC cells invasion and migration transfected with p50-pcDNA3.1 and pcDNA3.1 vector. ( * P < 0.05, ** P < 0.01).

Article Snippet: ROS in A549 and SK-MES-1 cells were evaluated by using the ROS assay kit (Elabscience, China).

Techniques: Activation Assay, Western Blot, Over Expression, Quantitative RT-PCR, Transfection, Plasmid Preparation, Migration

Journal: Langmuir

Article Title: Profiling High-Abundance Serum Proteins in the Corona of Nanodiamonds Using Mass Spectrometry

doi: 10.1021/acs.langmuir.5c06674

Figure Lengend Snippet: PC influences the cytotoxicity of NDs. (a) Bright field images of A549 cells before and after exposure to oxDND and HPHT ND, with and without PC. (b) Survival rate of A549 cells before and after exposure to NDs, with and without PC, measured using the CCK-8 assay ( n = 3; * p < 0.05).

Article Snippet: The viability of A549 cells exposed to NDs, with and without PC, was determined using the CCK-8 assay (Elabscience, Japan).

Techniques: CCK-8 Assay

Journal: Prostaglandins & other lipid mediators

Article Title: Diosgenin suppresses COX-2 and mPGES-1 via GR and improves LPS-induced liver injury in mouse.

doi: 10.1016/j.prostaglandins.2021.106580

Figure Lengend Snippet: Fig. 1. Suppression of cyclooxygenase (COX)-2 and microsomai prostaglandin E synthase-1 (mPGES-1) by diosgenin and recovery of COX-2 expression by glucocorticoid receptor (GR) antagonist via nuclear factor-kappa B (NF-κB) translocation. A: A549 cells were incubated with or without diosgenin (100 or 1,000 nM) or dexamethasone (Dex; 100 nM) for 48 h. The protein expression levels of COX-2 and mPGES-1 were analyzed by western blotting. The ratio was represented as a relative value against the control cells. B: AA549 cells were incubated with diosgenin (100 nM) for 24 h with a GR antagonist, RU486 (0–100 nM). The expression levels of PTGS2 (COX-2) were analyzed by quantitative RT-PCR and are shown as relative values against control cells without diosgenin, Dex, and RU486. Data are represented as the means ± S.E. of three separate experiments; p < 0.01 compared with *diosgenin (−)/Dex (−)/RU486 (−). C: Intracellular localization of NF-κB (red) and DAPI (blue) was visualized by CLSM. Scale bar: 20 μm.

Article Snippet: A549 cell lysates (6.6 × 105 cells) after the treatment, with or without diosgenin for 48 h, were subjected to western blotting using rabbit anti-COX-2 antibody (1:1,000, Novus Biochemicals LLC, Centennial, Co), rabbit anti-mPGES-1 antibody (1:250, Santa Cruz Biotechnology, Dallas, TX), or rabbit anti-β-actin antibody (1:1,000, Cell Signaling Technology, Boston, MA).

Techniques: Expressing, Translocation Assay, Incubation, Western Blot, Control, Quantitative RT-PCR

Journal: The European Respiratory Journal

Article Title: R othia mucilaginosa is an anti-inflammatory bacterium in the respiratory tract of patients with chronic lung disease

doi: 10.1183/13993003.01293-2021

Figure Lengend Snippet: Three-dimensional (3D) lung epithelial responses to pro-inflammatory stimuli in the presence and absence of members of the lung microbiota: Pseudomonas aeruginosa PAO1 (Pa), Rothia mucilaginosa DSM20746 (Rm), Staphylococcus aureus SP123 (S), Streptococcus anginosus LMG14696 (St), Achromobacter xylosoxidans LMG26680 (A) and Gemella haemolysans LMG18984 (G). a, b) Interleukin (IL)-8 production by 3D A549 cells after 4 h exposure to a) single bacterial cultures or b) co-cultures of various lung microbiota members with P. aeruginosa PAO1 at an MOI of 30:1. c) IL-8 production by 3D A549 cells after 4 or 24 h exposure to 100 μg·mL −1 lipopolysaccharide (LPS) alone or in co-culture with R. mucilaginosa at an MOI 10:1 (4 h) or 1:1 (24 h). d) IL-6, IL-8, granulocyte–macrophage colony-stimulating factor (GM-CSF) and monocyte chemoattractant protein (MCP)-1 production of 3D A549 cells after 4 h exposure to P. aeruginosa alone or in co-culture with R. mucilaginosa at an MOI of 10:1. NC: negative control (uninfected 3D epithelial cells in serum-free medium). Data represent mean± sem or mean, n≥3. *: p<0.05; **: p<0.001.

Article Snippet: A 3D epithelial cell model of NF-κB–luciferase-transfected A549 cells (BPS Bioscience, San Diego, CA, USA) was developed as described for the 3D A549 model.

Techniques: Co-Culture Assay, Negative Control

Journal: The European Respiratory Journal

Article Title: R othia mucilaginosa is an anti-inflammatory bacterium in the respiratory tract of patients with chronic lung disease

doi: 10.1183/13993003.01293-2021

Figure Lengend Snippet: Differential expression of genes involved in inflammation by three-dimensional (3D) A549 alveolar epithelial cells exposed to Pseudomonas aeruginosa versus P. aeruginosa in combination with Rothia mucilaginosa . Quantitative PCR analysis showing average fold changes in mRNA levels of 3D A549 cells stimulated by P. aeruginosa PAO1 versus a co-culture of P. aeruginosa and R. mucilaginosa . n=3. *: statistically significant (p<0.05) fold change.

Article Snippet: A 3D epithelial cell model of NF-κB–luciferase-transfected A549 cells (BPS Bioscience, San Diego, CA, USA) was developed as described for the 3D A549 model.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Co-Culture Assay

Journal: The European Respiratory Journal

Article Title: R othia mucilaginosa is an anti-inflammatory bacterium in the respiratory tract of patients with chronic lung disease

doi: 10.1183/13993003.01293-2021

Figure Lengend Snippet: Effect of Rothia mucilaginosa (Rm) on NF-κB pathway activation by Pseudomonas aeruginosa (Pa). a) Activation of the NF-κB pathway measured via luminescence of three-dimensional (3D) NF-κB reporter A549 cells. 3D cells were exposed for 4 h to P. aeruginosa PAO1 alone or in co-culture with R. mucilaginosa DSM20746. b) Semiquantitative determination (by Western blotting) of proteins ( i.e. A20, IκBα, p65 and phosphorylated (p)-IκBα) produced by 3D A549 cells stimulated with P. aeruginosa PAO1 with or without R. mucilaginosa DSM20746 for 15 min (15′), 30 min (30′), 1 h and 4 h. c) Band intensity (normalised to β-actin) of Western blot at 4 h of 3D A549 cells stimulated with P. aeruginosa PAO1 with or without R. mucilaginosa DSM20746. RLU: relative light units; NC: negative control (uninfected 3D NF-κB reporter A549 cells in serum-free GTSF-2 medium). Data represent mean± sem , n=3. *: p<0.05; **: p<0.01; ***: p<0.001.

Article Snippet: A 3D epithelial cell model of NF-κB–luciferase-transfected A549 cells (BPS Bioscience, San Diego, CA, USA) was developed as described for the 3D A549 model.

Techniques: Activation Assay, Co-Culture Assay, Western Blot, Produced, Negative Control

Journal: The European Respiratory Journal

Article Title: R othia mucilaginosa is an anti-inflammatory bacterium in the respiratory tract of patients with chronic lung disease

doi: 10.1183/13993003.01293-2021

Figure Lengend Snippet: The anti-inflammatory effect of Rothia mucilaginosa (Rm) is mediated by its cell-free supernatant. a) Interleukin (IL)-8 production by three-dimensional (3D) A549 epithelial cells after exposure to P. aeruginosa PAO1 (Pa) for 4 h, with or without R. mucilaginosa DSM20746 live cells, lysed cells or cell-free supernatant. b) Quantification of NF-κB pathway activation after 4 h exposure to lipopolysaccharide (LPS) (100 μg·mL −1 ) with or without R. mucilaginosa DSM20746 cell-free supernatant. The multiplicity of infection was 10:1 in all experiments. RLU: relative light units; NC: negative control (uninfected 3D epithelial cells). Data represent mean± sem , n≥3. ***: p<0.001.

Article Snippet: A 3D epithelial cell model of NF-κB–luciferase-transfected A549 cells (BPS Bioscience, San Diego, CA, USA) was developed as described for the 3D A549 model.

Techniques: Activation Assay, Infection, Negative Control