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: 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

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: OTUD6A expression is upregulated in airway epithelial cells in asthma. A‐B) Immunohistochemical (IHC) staining and quantification of OTUD6A in human lung tissues from healthy controls ( n = 2) and asthmatic patients ( n = 2). Scale bars: 100 µm. C‐D) Western blot analysis of OTUD6A expression in lung tissue from HDM‐CAM (C) and HDM‐AAM (D) mice. E‐F) Representative images of IHC for OTUD6A on mouse lung sections of HDM‐CAM and corresponding quantitative analysis ( n = 5). Scale bars: 100 µm. G‐H) Representative images of IHC for OTUD6A on mouse lung sections of HDM‐AAM and corresponding quantitative analysis ( n = 5). Scale bars; 100 µm. I‐J) Immunofluorescence staining for OTUD6A in the mouse lung tissue of HDM‐AAM and corresponding quantitative analysis ( n = 5). Scale bars: 50 µm. K) Western blot analysis of OTUD6A protein levels in BEAS‐2B cells stimulated with HDM (100 µg/mL) at indicated time points ( n = 5). L‐M) BEAS‐2B cells transfected with OTUD6A or control vector for 24 h. L) Western blot analysis of ZO‐1 and Occludin. M) RT‐qPCR analysis of IL‐25 , IL‐33 , and TSLP mRNA levels ( n = 5). N‐O) HBEpiC cells transfected with OTUD6A or control vector for 24 h. N) Western blot analysis of ZO‐1 and Occludin. O) ELISA analysis of IL‐33, IL‐25, and TSLP levels in cell supernatant ( n = 5). P‐Q) HBEpiC cells transfected with siOTUD6A or control vector for 48 h and stimulated with HDM (100 µg/mL) for 6 h. P) Western blot analysis of OTUD6A, ZO‐1, and Occludin. Q) ELISA analysis of IL‐33, IL‐25, and TSLP levels in cell supernatant ( n = 5). Data are presented as mean ± SEM. P values determined by two‐tailed unpaired t‐test or one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The human bronchial epithelial cell line BEAS‐2B (RRID: CVCL_0168, Strain: N/A, Clone number: N/A, Cat# STCC10202G) were purchased from Servicebio (Wuhan, China).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Western Blot, Immunofluorescence, Staining, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: OTUD6A promotes airway remodeling tvia PI3K/AKT‐EMT signaling. A) Serum TGFβ1 levels in HDM‐CAM mice ( n = 5). B‐C) Masson's trichrome staining and quantification of lung sections ( n = 5). Scale bars: 100 µm. D) Western blot analysis of Vimentin, TGFβ1, α‐SMA, and Fibronectin in lung tissues. E) RT‐qPCR analysis of Tgfb1 , Acta2 and Col1a1 mRNA levels ( n = 5). F) Western blot analysis of PI3K/AKT and EMT markers in HDM‐CAM lung tissues. G) Western blot analysis of PI3K/AKT, EMT markers, Vimentin, and Fibronectin in BEAS‐2B cells transfected with Flag‐OTUD6A for 24 h. H‐I) Western blot analysis of PI3K/AKT and EMT markers in BEAS‐2B cells transfected with siOTUD6A for 48 h and stimulated with HDM for 6 h. J) Immunofluorescence staining of E‐cadherin and N‐cadherin in BEAS‐2B cells transfected with Flag‐OTUD6A for 24 h. Scale bars: 50 µm. K‐N) HBEpiC cells transfected with siOTUD6A or negative control for 48 h and stimulated with HDM (100 µg/mL) for 6 h. Western blot analysis of PI3K/AKT and EMT markers. Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The human bronchial epithelial cell line BEAS‐2B (RRID: CVCL_0168, Strain: N/A, Clone number: N/A, Cat# STCC10202G) were purchased from Servicebio (Wuhan, China).

Techniques: Staining, Western Blot, Quantitative RT-PCR, Transfection, Immunofluorescence, Negative Control

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: hResistin/mRELMα is a potential substrate of OTUD6A. A) Multi‐omics identification of OTUD6A substrates. B‐C) Western blot of mRELMα expression in HDM‐CAM (B) and HDM‐AAM (C) lung tissues. D‐E) IHC staining of mRELMα in HDM‐CAM (D) and HDM‐AAM (E) lung tissues. Scale bars: 100 µm. F‐G) Correlation analysis of mRELMα and OTUD6A IHC staining in HDM‐CAM (F) and HDM‐AAM (G). Two random fields per mouse. H) Western blot analysis of hResistin in BEAS‐2B cells transfected with Flag‐OTUD6A and control vector for 24 h. I) Western blot analysis of hResistin in HBEpiC cells transfected with Flag‐OTUD6A and control vector for 24 h. J) Western blot analysis of hResistin in BEAS‐2B cells transfected with siOTUD6A for 24 h with or without HDM (100 µg/mL) treatment for 6 h. K) Western blot analysis of hResistin in HBEpiC cells transfected with siOTUD6A for 24 h with or without HDM (100 µg/mL) treatment for 6 h. Data are presented as mean ± SEM. P values determined by two‐tailed unpaired t‐test or one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The human bronchial epithelial cell line BEAS‐2B (RRID: CVCL_0168, Strain: N/A, Clone number: N/A, Cat# STCC10202G) were purchased from Servicebio (Wuhan, China).

Techniques: Biomarker Discovery, Western Blot, Expressing, Immunohistochemistry, Transfection, Control, Plasmid Preparation, Two Tailed Test

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: hResistin/PI3K mediates OTUD6A‐induced EMT process and the expression of epithelial‐derived alarmins. A‐G) BEAS‐2B cells transfected with siResistin for 48 h and then transfected with Flag‐OTUD6A for 24 h. A) RT‐qPCR analysis of TSLP , IL‐25 , IL‐33 mRNA level in BEAS‐2B cells ( n = 5). B) ELISA analysis of TSLP, IL‐25, and IL‐33 levels in cell supernatant ( n = 5). C) RT‐qPCR analysis of TGFB1 , ACTA2 , COL1A1 mRNA level in BEAS‐2B cells ( n = 5). D‐E) Western blot analysis of OTUD6A, hResistin, EMT, and PI3K/AKT markers. F) Immunofluorescence staining of E‐cadherin and N‐cadherin. Scale bars: 50 µm. G) Cell wound healing assay. Scale bars: 50 µm. H‐K) BEAS‐2B cells were pretreated with LY294002 (30 µ m ) for 30 min and transfected with Flag‐OTUD6A for 24 h. H) Western blot analysis of hResistin and EMT markers. I‐K) ELISA analysis of IL‐33, IL‐25, and TSLP levels in cell supernatant ( n = 5). Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The human bronchial epithelial cell line BEAS‐2B (RRID: CVCL_0168, Strain: N/A, Clone number: N/A, Cat# STCC10202G) were purchased from Servicebio (Wuhan, China).

Techniques: Expressing, Derivative Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, Wound Healing Assay

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: OTUD6A deubiquitinates hResistin in the endoplasmic reticulum to divert it from proteasomal degradation to secretion. A‐B) Co‐immunoprecipitation of endogenous OTUD6A and mRELMα in HDM‐CAM (A) and HDM‐AAM (B) lung tissues. C) Co‐immunoprecipitation of exogenous OTUD6A and hResistin in BEAS‐2B cells expressed Flag‐OTUD6A and HA‐hResistin. D) Immunofluorescence of hResistin and OTUD6A in BEAS‐2B cells transfected with Flag‐OTUD6A and HA‐hResistin. Scale bars: 20 µm. E) CHX chase assay for hResistin stability ( n = 3). F) BEAS‐2B cells were stimulated with DMSO, MG132 (10 µ m ), Epoxomicin (2.5 µ m ), BTZ (5 µ m ), CQ (25 µ m ), 3‐MA (10 m m ), Bafilomycin A1 (1 µ m ), or Concanamycin A (0.5 µ m ) for 6 h. Western blot analysis of hResistin in BEAS‐2B cells. G) HA‐hResistin, Flag‐OTUD6A, and Myc‐Ub were transfected into BEAS‐2B cells and then subjected to 10 µ m MG132 for 6 h. Ubiquitinated hResistin was detected. H) HA‐hResistin, Flag‐OTUD6A, Myc‐WT Ub, Myc‐K48 Ub, and Myc‐K63 Ub were transfected into BEAS‐2B cells and then subjected to 10 µ m MG132 for 6 h. Ubiquitinated hResistin was detected. I) BEAS‐2B cells were stimulated with DMSO, MG132 (10 µ m ), Epoxomicin (2.5 µ m ), or BTZ (5 µ m ) for 6 h or transfected with Flag‐OTUD6A for 24 h, ELISA analysis of hResistin levels in cell supernatant ( n = 5). J) BEAS‐2B cells transfected with Myc‐K48 Ub or Myc‐K63 Ub for 24 h, ELISA analysis of hResistin levels in cell supernatant ( n = 5). K) Immunofluorescence of Resistin and Calnexin in BEAS‐2B cells treated with MG132 (10 µ m ) or not for 6 h. Scale bars: 50 µm. L) Immunofluorescence of Resistin and OTUD6A in BEAS‐2B cells treated with MG132 (10 µ m ) or not for 6 h. Scale bars: 50 µm. Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The human bronchial epithelial cell line BEAS‐2B (RRID: CVCL_0168, Strain: N/A, Clone number: N/A, Cat# STCC10202G) were purchased from Servicebio (Wuhan, China).

Techniques: Immunoprecipitation, Immunofluorescence, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: OTUD6A undergoes deubiquitination at K2 and K19 of hResistin through its OTU domain and C152 residue. A) Schematic diagram of OTUD6A and its truncated mutants. B) Flag‐OTUD6A or its truncated mutants and HA‐hResistin transfected into BEAS‐2B cells. Mapping of OTUD6A‐hResistin interaction domains. C) Conserved catalytic cysteine (C152A) in OTUD6A orthologs. D) BEAS‐2B cells transfected with Flag‐OTUD6A (WT or C152A), following CHX (100 µg/mL) treatment for the indicated times. CHX chase assay for hResistin stability. E) HA‐hResistin and Myc‐Ub were transfected into BEAS‐2B cells together with Flag‐OTUD6A (WT or C152A) and then subjected to 10 µ m MG132 for 6 h. Ubiquitinated hResistin was detected. F) HA‐hResistin was transfected into BEAS‐2B cells together with Flag‐OTUD6A (WT or C152A) and then subjected to CHX (100 µg/mL) for 6 h. ELISA analysis of hResistin levels in cell supernatant ( n = 5). G‐H) HA‐hResistin (WT, K2R, K19R or K2/K19R), Flag‐OTUD6A, and Myc‐Ub were transfected into BEAS‐2B cells and then subjected to 10 µ m MG132 for 6 h. Ubiquitinated hResistin was detected. I‐J) HA‐hResistin (K2/K19R) and Flag‐OTUD6A (WT or C152A) were transfected into BEAS‐2B cells for 24 h, and then subjected to CHX (100 µg/mL) for 6 h. I) Western blot analysis of hResistin in BEAS‐2B cells. J) ELISA analysis of hResistin levels in cell supernatant ( n = 5). Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The human bronchial epithelial cell line BEAS‐2B (RRID: CVCL_0168, Strain: N/A, Clone number: N/A, Cat# STCC10202G) were purchased from Servicebio (Wuhan, China).

Techniques: Residue, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot