Journal: eLife

Article Title: Suppressor of cytokine signaling (SOCS)5 ameliorates influenza infection via inhibition of EGFR signaling

doi: 10.7554/eLife.20444

Figure Lengend Snippet: ( A ) Primary mouse airway epithelial cells (mAEC) from wild-type and Socs5 −/− mice were purified and cultured in vitro for 7 days prior to infection with influenza virus H1N1 PR8. ( B ) 8 h post-infection cell monolayers were fixed and stained by immunofluorescence for detection of viral nuclear protein. Results are shown as the mean % of infected cells from four technical replicates and are representative of 2 experiments. ( C ) mAEC were infected with PR8 (MOI 1) and then incubated in the presence of trypsin for 2, 8, 24 and 48 h. Culture supernatants were analyzed for infectious virus by plaque assay. Results are shown as the fold increase over the level of virus present at 2 h post-infection. Mean ± S.E.M. are shown for three technical replicates and are representative of 2 experiments. ( D ) mAECs were infected (MOI 5) and cytokine levels in culture supernatants measured by Bioplex at 24 h post-infection. Individual and mean values are shown for technical replicates derived from purified cells pooled from five mice and are representative of 2 experiments. *p<0.05. DOI: http://dx.doi.org/10.7554/eLife.20444.009

Article Snippet: The minimally immortalised bronchial epithelial cell line (BCi-NS1.1; ) was cultured in BEBM complete media (Lonza) and was obtained from R.G.

Techniques: Purification, Cell Culture, In Vitro, Infection, Virus, Staining, Immunofluorescence, Incubation, Plaque Assay, Derivative Assay

Journal: eLife

Article Title: Suppressor of cytokine signaling (SOCS)5 ameliorates influenza infection via inhibition of EGFR signaling

doi: 10.7554/eLife.20444

Figure Lengend Snippet: Primary human airway epithelial cells (hAECs) from healthy individuals, smokers with COPD and smokers without lung disease, were cultured as described and infected with H3N2, H1N1 and H11N9 influenza virus strains (MOI 5). ( A ) 24 h post-infection Socs5 mRNA was measured by Q-PCR and is shown as the fold-change from a media only control. Mean ± S.E.M.; n = 3. ( B ) Data for healthy controls shown in ( A ) is plotted against the expression of Socs5 mRNA following infection with H5N1 (MOI 0.005). *p<0.05 Infection vs Media, + p<0.05 healthy vs COPD and H5N1 vs other subtypes/strains; Mean ± S.E.M.; n = 3. ( C–F ) hAECs from healthy individuals and COPD patients were cultured and SOCS5 depleted using siRNA ( C,E ). Alternatively, SOCS5 expression was increased in hAECs by transfection with SOCS5 cDNA (SOCS5 vector) ( D,F ) prior to infection with H1N1 (MOI 5). Cells were lysed at 24 h ( C,D ) or 2 h ( E,F ) post-infection and analyzed by immunoblotting with the indicated antibodies. DOI: http://dx.doi.org/10.7554/eLife.20444.013

Article Snippet: The minimally immortalised bronchial epithelial cell line (BCi-NS1.1; ) was cultured in BEBM complete media (Lonza) and was obtained from R.G.

Techniques: Cell Culture, Infection, Virus, Control, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: eLife

Article Title: Suppressor of cytokine signaling (SOCS)5 ameliorates influenza infection via inhibition of EGFR signaling

doi: 10.7554/eLife.20444

Figure Lengend Snippet: ( A ) Human primary airway epithelial cells (hAECs) from healthy individuals (Healthy), smokers with COPD and smokers without lung disease (Smoker), were cultured and infected with influenza virus H1N1 (MOI 5). ( A–D ) Cells were lysed at 2 h post-infection and analyzed by Western blotting with the indicated antibodies. ( B ) Densitometric values were derived by first normalizing to GAPDH loading controls and are expressed as fold-change from healthy untreated controls (Media). *p<0.05 H1N1 vs media control, + p<0.05 COPD vs healthy; Mean ± S.E.M.; n = 5. ( C ) hAECs from healthy individuals were cultured and transfected with either siRNA to deplete Socs5 (SOCS5 -si ), control siRNA (scrambled) or vehicle alone. ( D ) Alternatively, SOCS5 expression was increased in hAECs by transfection with cDNA encoding SOCS5 (SOCS5), empty vector or vehicle alone, prior to infection. DOI: http://dx.doi.org/10.7554/eLife.20444.014

Article Snippet: The minimally immortalised bronchial epithelial cell line (BCi-NS1.1; ) was cultured in BEBM complete media (Lonza) and was obtained from R.G.

Techniques: Cell Culture, Infection, Virus, Western Blot, Derivative Assay, Control, Transfection, Expressing, Plasmid Preparation

Journal: eLife

Article Title: Suppressor of cytokine signaling (SOCS)5 ameliorates influenza infection via inhibition of EGFR signaling

doi: 10.7554/eLife.20444

Figure Lengend Snippet: ( A ) Mice were infected i.n. with 35 pfu influenza virus H1N1 PR8 and viral titres determined in lung homogenates 24 h post-PR8 infection, by plaque assay. Mice were treated with vehicle (captisol), PI3K inhibitor (BKM-120) or EGFR inhibitor (Erlotinib) 4 h post-PR8 inoculation. ( B ) Primary mouse airway epithelial cells (mAEC) from wild-type and Socs5 −/− mice were purified and cultured in vitro for 7 days prior to infection with influenza virus H1N1 PR8 (MOI 1) and then incubated in the presence of trypsin for 24 h. PI3K (BKM-120) and EGFR inhibitors (AG1478) were included in the culture media during and post-infection. Culture supernatants were analyzed for infectious virus by plaque assay. ( C ) Primary human airway epithelial cells (hAECs) from healthy individuals were cultured as described and infected with H1N1 strain (A/Auckland/1/2009; MOI 5). PI3K inhibitor (Wortmannin) and EGFR inhibitor (Erlotinib) were added 3 h prior to virus inoculation. Culture supernatants were analyzed for infectious virus by plaque assay 24 h post-infection. *p<0.05, **<0.005. ( A – C ; Mean ± S.E.M of indicated n ). p values were determined by ( A,B ) unpaired student’s t-test or ( C ) a Mann-Whitney U test. DOI: http://dx.doi.org/10.7554/eLife.20444.019

Article Snippet: The minimally immortalised bronchial epithelial cell line (BCi-NS1.1; ) was cultured in BEBM complete media (Lonza) and was obtained from R.G.

Techniques: Infection, Virus, Plaque Assay, Purification, Cell Culture, In Vitro, Incubation, MANN-WHITNEY

Journal: eLife

Article Title: Suppressor of cytokine signaling (SOCS)5 ameliorates influenza infection via inhibition of EGFR signaling

doi: 10.7554/eLife.20444

Figure Lengend Snippet: The minimally immortalised bronchial epithelial cell line (BCi-NS1.1) was cultured in BEBM complete media (Lonza) and transfected with control siRNA or siRNA to deplete Socs5 , prior to addition of the proteasomal inhibitor MG132 (10 μM; 4 h). Cells were then infected with influenza virus H1N1 (MOI 5) and lysed 2 h post-infection. Immunoblot analysis with the indicated antibodies. DOI: http://dx.doi.org/10.7554/eLife.20444.017

Article Snippet: The minimally immortalised bronchial epithelial cell line (BCi-NS1.1; ) was cultured in BEBM complete media (Lonza) and was obtained from R.G.

Techniques: Cell Culture, Transfection, Control, Infection, Virus, Western Blot

Journal: Purinergic Signalling

Article Title: Autocrine signaling via release of ATP and activation of P2X7 receptor influences motile activity of human lung cancer cells

doi: 10.1007/s11302-014-9411-x

Figure Lengend Snippet: TGF-β1-induced migration is mediated by release of ATP and activation of P2X7 receptor in H292 lung cancer cells, but not BEAS-2B normal lung cells. a The expression of P2X7 receptor in BEAS-2B, A549, PC-9, and H292 cells was detected by immunoblotting as described in “Materials and methods”. b, c BEAS-2B cells (b) or H292 cells (c) were stimulated with TGF-β1 (5 ng/mL) and incubated for the indicated times, then the concentration of ATP in the culture medium was measured as described in “Materials and methods”. d, e Cell migration was examined by means of Transwell assay as described in “Materials and methods”. BEAS-2B cells (d) or H292 cells (e) were pretreated for 30 min with apyrase (20 U/mL) or A438079 (100 μM) and then stimulated for 24 h with TGF-β1 (5 ng/mL). The lower membrane surfaces were photographed through a microscope at ×20 magnification, and migrated cells in each field were counted. Values are means ± SE (n = 4–10). A significant difference between the indicated group and control group is indicated by *** or ### (P < 0.001), ** (P < 0.01), †(P < 0.05)

Article Snippet: Cell culture BEAS-2B normal human bronchial epithelial cell line and NCI-H292 human lung mucoepidermoid carcinoma cell line were purchased from DS Pharma Biomedical (Osaka, Japan).

Techniques: Migration, Activation Assay, Expressing, Western Blot, Incubation, Concentration Assay, Transwell Assay, Microscopy

Journal: Dose-Response

Article Title: LncRNA MIR210HG Facilitates Non-Small Cell Lung Cancer Progression Through Directly Regulation of miR-874/STAT3 Axis

doi: 10.1177/1559325820918052

Figure Lengend Snippet: The expression of miR210HG increases in NSCLC samples and cells. A, the relative expression of miR210HG was detected in NSCLC tissues and paired normal samples. B, The relative expression of miR210HG was detected in NSCLC cells compared with the normal human bronchial epithelial cell line 16HBE. * P <.05, ** P < .01. miR210HG indicates micro RNA; NSCLC, non-small cell lung cancer.

Article Snippet: Four human NSCLC cell lines (NCI-H1975, H1299, A549, and GLC-82) and the normal human bronchial epithelial cell line 16HBE were purchased from ScienCell Research Laboratories (Carlsbad, California) and cultured in RPMI 1640 medium (Gibco, Carlsbad, California) with 10% fetal bovine serum (FBS; Gibco, Grand Island, New York) at 37°C with 5% CO 2 .

Techniques: Expressing

Journal: Journal of Virology

Article Title: Discovery and Evaluation of Entry Inhibitors for SARS-CoV-2 and Its Emerging Variants

doi: 10.1128/JVI.01437-21

Figure Lengend Snippet: Measurement of cytotoxicity of five drug-like compounds using MTT assay. Measurement of cytotoxicity of five drug-like compounds. (A–E) Viability of HEK293T-hACE2 cells in the presence of an indicated concentration of the compounds. (F, G) Measurement of cytotoxicity of MU-UNMC-1 and MU-UNMC-2 in Vero-STAT1 KO cells in the presence of an indicated concentration of the compounds. (H, I) Measurement of cytotoxicity of MU-UNMC-1 and MU-UNMC-2 in UNCN1T cells in the presence of an indicated concentration of the compounds. (J, K) Measurement of cytotoxicity of MU-UNMC-1 and MU-UNMC-2 in Calu-3 cells in the presence of an indicated concentration of the compounds.

Article Snippet: UNCN1T cells (a human bronchial epithelial cell line; Kerafast catalog number ENC011) were cultured in BEGM media (Bronchial Epithelial Cell Growth Medium; Lonza catalog number CC-3170) in FNC (Athena Enzyme Systems catalog number 0407) coated 96-well plates.

Techniques: MTT Assay, Concentration Assay

Journal: Journal of Virology

Article Title: Discovery and Evaluation of Entry Inhibitors for SARS-CoV-2 and Its Emerging Variants

doi: 10.1128/JVI.01437-21

Figure Lengend Snippet: SARS-CoV-2 dose-response curve in MU-UNMC-1 and MU-UNMC-2 treated and SARS-CoV-2 infected UNCN1T and Vero-STAT1 knockout cells. (A, B) MU-UNMC-1 (in blue) and MU-UNMC-2 (in green) dose-response curve by percentage inhibition of SARS-CoV-2 replication 24 and 48 hpi in UNCN1T cells with indicated drug concentrations. (C, D) MU-UNMC-1 (in blue) and MU-UNMC-2 (in green) dose-response curve by percentage inhibition of SARS-CoV-2 replication 24 and 48 hpi in Vero-STAT1 knockout cells with indicated compound concentrations.

Article Snippet: UNCN1T cells (a human bronchial epithelial cell line; Kerafast catalog number ENC011) were cultured in BEGM media (Bronchial Epithelial Cell Growth Medium; Lonza catalog number CC-3170) in FNC (Athena Enzyme Systems catalog number 0407) coated 96-well plates.

Techniques: Infection, Knock-Out, Inhibition

Journal: Journal of Virology

Article Title: Discovery and Evaluation of Entry Inhibitors for SARS-CoV-2 and Its Emerging Variants

doi: 10.1128/JVI.01437-21

Figure Lengend Snippet: Combinational effect of remdesivir and MU-UNMC-1 treatment against SARS-CoV-2 infected UNCN1T cells at 24 h postinfection. (A) Dose response curve of remdesivir in SARS-CoV-2 infected UNCN1T cells at 24 hpi in the presence of different fixed concentrations of MU-UNMC-1; (B) Dose-response curve of MU-UNMC-1 in SARS-CoV-2 infected UNCN1T cells at 24 hpi in the presence of a different fixed concentration of remdesivir; (C) Dose-response percent inhibition matrix of single and combined treatment of remdesivir and MU-UNMC-1 in SARS-CoV-2 infected UNCN1T cells at 24 hpi. (D) 3-D interaction landscape between remdesivir and MU-UNMC-2 calculated based on Loewe additive model using SynergyFinder v.2 in SARS-CoV-2 infected UNCN1T cells at 24 hpi (Loewe synergy score -30.69; with most synergistic area score of -21.34).

Article Snippet: UNCN1T cells (a human bronchial epithelial cell line; Kerafast catalog number ENC011) were cultured in BEGM media (Bronchial Epithelial Cell Growth Medium; Lonza catalog number CC-3170) in FNC (Athena Enzyme Systems catalog number 0407) coated 96-well plates.

Techniques: Infection, Concentration Assay, Inhibition

Journal: Journal of Virology

Article Title: Discovery and Evaluation of Entry Inhibitors for SARS-CoV-2 and Its Emerging Variants

doi: 10.1128/JVI.01437-21

Figure Lengend Snippet: Combinational effect of remdesivir and MU-UNMC-2 treatment against SARS-CoV-2 infected UNCN1T cells at 24 h postinfection. (A) Dose-response curve of remdesivir in SARS-CoV-2 infected UNCN1T cells at 24 hpi in the presence of a different fixed concentration of MU-UNMC-2; (B) dose-response curve of MU-UNMC-2 in SARS-CoV-2 infected UNCN1T cells at 24 hpi in the presence of a different fixed concentration of remdesivir; (C) dose-response percent inhibition matrix of single and combined treatment of remdesivir and MU-UNMC-2 in SARS-CoV-2 infected UNCN1T cells at 24 hpi. (D) 3-D interaction landscape between remdesivir and MU-UNMC-2 calculated based on Loewe additive model using SynergyFinder v.2 in SARS-CoV-2 infected UNCN1T cells at 24 hpi (Loewe synergy score 26.63; with most synergistic area score of 37.25).

Article Snippet: UNCN1T cells (a human bronchial epithelial cell line; Kerafast catalog number ENC011) were cultured in BEGM media (Bronchial Epithelial Cell Growth Medium; Lonza catalog number CC-3170) in FNC (Athena Enzyme Systems catalog number 0407) coated 96-well plates.

Techniques: Infection, Concentration Assay, Inhibition

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Long non-coding RNA NKILA inhibits proliferation and migration of lung cancer via IL-11/STAT3 signaling

doi:

Figure Lengend Snippet: The expression of NKILA was higher in normal human bronchial epithelial cell line. For (A) and (B), the expression level of lncRNA-NKILA was analyzed by Q-PCR. The expression of NKILA was analyzed in NSCLC tissues (C), Kaplan-Meier analyses of the correlations between lncRNA-NKILA expression level and survival (D). Values are means ± SEM for n = 7-8. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

Article Snippet: Cell culture A normal human bronchial epithelial cell line (16HBE) and NSCLC adenocarcinoma cell lines (A549, NCI-H1975) were purchased from the Biopike Biological company.

Techniques: Expressing, Control

Journal: Cell Medicine

Article Title: A Rotating Bioreactor for Scalable Culture and Differentiation of Respiratory Epithelium

doi: 10.3727/215517914X681794

Figure Lengend Snippet: Schematic showing setup and culture of constructs within bioreactor. Normal human bronchial epithelial (NHBE) cells are placed on the collagen-coated polytetrafluorethylene (PTFE) membrane bonded to an absorbent polyvinylidene fluoride (PVDF) sponge layer and allowed to adhere (A). (B and C) Scanning electron micrographs of the two construct layers, pictured without cells, showing their relative porosity and structure. Scale bars: 2 µm. This construct is rolled into a cylinder and placed inside the reactor vial where it aligns with the walls. The reactor is sealed, with ports to allow gas and media exchange (D). The reactor vial is half filled with media and rolled continuously throughout culture, generating intermittent air–liquid interface (ALI) and submerged conditions (E). When ready for cell harvesting and analysis, the construct is removed from the reactor, and the upper PTFE membrane with attached cells is processed (F).

Article Snippet: NHBE Cell Culture B-ALI-certified normal human bronchial/tracheal epithelial (NHBE) cells, from a 35-year-old male, were obtained from Lonza (CC-2540S; Lonza Group Ltd., Basel, Switzerland).

Techniques: Construct, Membrane, Cell Harvesting

Journal: Cell Medicine

Article Title: A Rotating Bioreactor for Scalable Culture and Differentiation of Respiratory Epithelium

doi: 10.3727/215517914X681794

Figure Lengend Snippet: Tight junction formation. Images showing staining for zonula occludens-1 (ZO-1; red) tight junctions in static control and rotating ALI cultures (A–F). Note the initial formation of a tight junction network in static cultures and subsequent deterioration by day 21 (A–C) compared to the steady increase in junction density and organization over time in the rotating ALI cultures (D–F). Nuclei are stained with DAPI (blue). Quantitative assessment of cell ZO-1 expression from immunostaining confirmed this trend and is expressed as percentage of cells staining positive, with 95% confidence intervals (G). Expression of ZO-1 at day 14 and day 21 in NHBE cells cultured in rotating ALI culture system compared to static (submerged) controls (H).

Article Snippet: NHBE Cell Culture B-ALI-certified normal human bronchial/tracheal epithelial (NHBE) cells, from a 35-year-old male, were obtained from Lonza (CC-2540S; Lonza Group Ltd., Basel, Switzerland).

Techniques: Staining, Control, Expressing, Immunostaining, Cell Culture

Journal: Cell Medicine

Article Title: A Rotating Bioreactor for Scalable Culture and Differentiation of Respiratory Epithelium

doi: 10.3727/215517914X681794

Figure Lengend Snippet: Mucin 5AC expression. Representative images of mucin 5AC expression (red) for each condition at 7, 14, and 21 days of culture (A–F). Both experimental conditions showed an increase in expression over time, but the rate was significantly higher for the rotating ALI cultures, as can be seen by comparing (C) and (F). Nuclei are stained with DAPI (blue). Quantitative measure of mucin expression over time for both experimental conditions, expressed as percentage of cells staining positive, with 95% confidence intervals (G). RT-PCR analysis of mucin 5AC expression in NHBE cells cultured in rotating ALI system at day 14 and day 21 compared to the cells cultured in static conditions (H).

Article Snippet: NHBE Cell Culture B-ALI-certified normal human bronchial/tracheal epithelial (NHBE) cells, from a 35-year-old male, were obtained from Lonza (CC-2540S; Lonza Group Ltd., Basel, Switzerland).

Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: Cell Medicine

Article Title: A Rotating Bioreactor for Scalable Culture and Differentiation of Respiratory Epithelium

doi: 10.3727/215517914X681794

Figure Lengend Snippet: Cilia development. Immunocytochemistry for each experimental condition across all relevant time points (A–F). Acetylated tubulin can be found in the cytoplasm of nonpolarized NHBE cells (red; A, D). This expression disappears after 2 weeks of culture in the differentiation-inducing media (B, C). Fully formed cilia stain strongly for acetylated tubulin, as can be seen in (E, F). Nuclei are stained with DAPI (blue). Scanning electron micrographs comparing the highest degree of ciliation observed in the day 21 static controls (G) and in the day 21 rotating ALI cultures (H). Forkhead box J1 (FOXJ1) gene expression in NHBE cells cultured in rotating ALI system at day 14 and day 21 compared to the cells cultured under static conditions (I). Scale bars: 50 µm (A–F); 5 µm and 2 µm (G and H), respectively.

Article Snippet: NHBE Cell Culture B-ALI-certified normal human bronchial/tracheal epithelial (NHBE) cells, from a 35-year-old male, were obtained from Lonza (CC-2540S; Lonza Group Ltd., Basel, Switzerland).

Techniques: Immunocytochemistry, Expressing, Staining, Gene Expression, Cell Culture

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicles enhance pulmonary transduction of stably associated adeno‐associated virus following intratracheal administration

doi: 10.1002/jev2.12324

Figure Lengend Snippet: EVAAV6 exhibits efficient penetration through human airway mucus and enhanced transduction of human bronchial epithelial (HBE) cell line. (a) Median MSD values of EVs and EVAAV6 in sputum samples spontaneously expectorated by CF patients. MSD is a square of distance traveled by an individual particulate matter within a predetermined time interval (i.e., time scale; τ = 1 s) and thus is directly proportional to the particle diffusion rate. The red dashed line indicates the MSD value of AAV6 previously measured in CF sputum (Gyorgy et al., ). respectively. n.s.: no significance (two‐tailed Student's t ‐test) (b) Luciferase activity measured in lysates of HBE cells (16HBE14o‐) treated with EVs, EVAAV6 or EV+AAV6. n.s.: no significance, **** p < 0.0001 (one‐way ANOVA).

Article Snippet: Human bronchial epithelial (HBE) cell line (16HBE14o‐) was a kind gift from Dr. Garry Cutting at Johns Hopkins University School of Medicine (Baltimore, MD, USA).

Techniques: Transduction, Diffusion-based Assay, Two Tailed Test, Luciferase, Activity Assay