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: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (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 knockout alleviates asthma in HDM‐induced acute asthma model. A) Schematic diagram depicting the procedure of HDM‐AAM. B) AHR assessed via acetylcholine challenge ( n = 5). C) Serum IgE levels measured by ELISA ( n = 5). D) Representative H&E and PAS staining of lung tissue. Scale bars: 100 µm. E) Quantification of bronchial epithelial thickness ( n = 5). F) BALF eosinophil counts determined by Wright‐Giemsa staining ( n = 5). G‐J) IL‐5 and IL‐13 levels in BALF (G, H) and lung homogenates (I, J) measured by ELISA ( n = 5). K‐N) RT‐qPCR analysis of Il5 , Il13 , Muc5ac , and Muc5b mRNA levels in lung tissue ( n = 5). O) ELISA analysis of IL‐25, IL‐33, and TSLP levels in BALF ( n = 5). P) Western blot and quantification of ZO‐1 and Occludin in lung tissues. Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Quantitative RT-PCR, Western Blot

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 knockout mitigates asthma in HDM‐induced chronic asthma model. A) Schematic diagram depicting the procedure of HDM‐CAM. B‐D) Rn, Ers, and Rrs assessed via methacholine challenge ( n = 5). E) Representative H&E staining of lung tissue. Scale bars: 100 µm. F) Serum IgE levels measured by ELISA ( n = 5). G‐H) Total cell counts (G) and protein concentration (H) in BALF ( n = 5). I‐L) IL‐5 and IL‐13 levels in BALF (I, J) and lung homogenates (K, L) measured by ELISA ( n = 5). M‐R) RT‐qPCR analysis of Il5 , Il13 , Muc5ac , Tslp , Il25 , and Il33 mRNA level in lung tissues ( n = 5). S‐U) ELISA analysis of IL‐25, IL‐33, and TSLP levels in BALF ( 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: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Knock-Out, Staining, Enzyme-linked Immunosorbent Assay, Protein Concentration, Quantitative RT-PCR

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: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (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: Lung‐specific OTUD6A knockdown attenuates HDM‐induced asthma. A) Schematic diagram depicting the procedure of HDM‐induced chronic asthma model. B) Western blot analysis of OTUD6A and mRELMα in lung tissues. C) AHR assessed via acetylcholine challenge ( n = 5). D) H&E staining of lung tissues. Scale bars: 100 µm. E) Serum IgE levels measured by ELISA ( n = 5). F‐G) Total cell counts (F) and protein concentration (G) in BALF ( n = 5). H‐I) IL‐5 (H) and IL‐13 (I) levels in BALF measured by ELISA ( n = 5). J‐L) RT‐qPCR analysis of Il4, Il5 , and Muc5ac mRNA levels in lung tissues ( n = 5). M) ELISA analysis of IL‐25, IL‐33, and TSLP levels in BALF (n = 5). N) Serum TGF‐β1 levels measured by ELISA ( n = 5). O) Masson's trichrome staining of lung sections. Scale bars: 100 µm. P) RT‐qPCR analysis of Tgfb1 , Acta2 , and Col1a1 mRNA levels in lung tissues ( n = 5). Q) Western blot analysis of Vimentin, TGFβ1, α‐SMA, and Fibronectin in the lung tissues. R) Western blot analysis of PI3K/AKT in the lung tissues. S) Western blot analysis of EMT markers in the lung tissues. Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Knockdown, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Protein Concentration, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 1. RAW cells chemotax toward CX3CL1 (fractalkine). A, Live cells were incubated with rabbit anti-CX3CR1 Ab or control IgG at 4°C before fixation and incubation with anti-rabbit IgG Ab conjugated to Alexa Fluor 488. Immunofluores- cence images are shown as well as the corresponding phase contrast im- ages (see insets). Scale bar 10 m. B, Cell migration in response to in- creasing doses of CX3CL1 was mea- sured as described in Materials and Methods. C, To distinguish between chemotaxis and chemokinesis, cell migration was assessed in response to 50 ng/ml CX3CL1 added in either the bottom chamber or the top chamber of the transmigration apparatus. D, The specificity of CX3CL1-induced chemotaxis was verified by preincu- bating the cells with 10 g/ml CX3CR1 neutralizing Ab or control IgG for 1 h before subjecting the cells to the transmigration assay. E, Cells were preincubated for 5 h with or without 250 ng/ml pertussis toxin (PTX) before measuring cell migra- tion as described above. n 3. , p 0.05 compared with the correspond- ing controls.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Incubation, Control, Migration, Chemotaxis Assay, Transmigration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 2. CX3CL1 treatment induces a rearrangement of the actin cytoskeleton. A, RAW cells were either untreated (Untr) or treated with 50 ng/ml CX3CL1 for 1 min before fixation and staining of F-actin using phalloidin conjugated to Alexa Fluor 568. Images are representatives of at least five independent experiments. Scale bar 10 m. B, Cells were treated for various times with CX3CL1, then fixed, and total F-actin, nor- malized to the cell number, was quantitatively measured as described in Materials and Methods. n 3–7 independent determinations for each time point.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 3. CX3CL1 stimulates tyrosine phosphorylation. A, RAW cells were treated for various times with 50 ng/ml CX3CL1, and total cell lysates were subjected to Western blotting using a phosphotyrosine-spe- cific Ab (PY) or using anti--actin Ab as a verification of equal protein loading. A Western blot representative of three independent experiments is shown. B, RAW cells were either untreated or treated with CX3CL1 for 1 min before fixation and costaining of F-actin and phosphotyrosine-contain- ing proteins. C, Murine bone marrow-derived macrophages, prepared as described in Materials and Methods, were stimulated with CX3CL1 for 1 min before costaining as shown for RAW cells. Unless otherwise noted, all images shown are z-sections collected 2.5 m above the cell-substratum interface (cell midsections) using a confocal microscope. Images noted top represent the same cells as those shown below, but were taken 5 m above the coverslip level to focus on dorsal ruffles. All images are representative of at least three independent experiments. Scale bars 10 m.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Phospho-proteomics, Western Blot, Derivative Assay, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 4. Syk is activated upon CX3CL1 treatment, and piceatannol inhibits CX3CL1-induced cell migration. A, RAW cells were either un- treated () or treated () with 50 ng/ml CX3CL1 for 1 min before lysis and immunoprecipitation of Syk (IP Syk) or phosphotyrosine-containing proteins (IP PY) using specific Abs. Samples were then subjected to West- ern blotting (WB) using the indicated Abs. The phosphospecific Ab used (P-Syk) specifically recognizes activated Syk (phosphorylated on residues Y519/520). Input represents the amount of Syk present in total cell lysates for each condition. Western blots representative of three independent ex- periments are shown. B, Cells were pretreated with 50 m piceatannol (Syk inhibitor) or DMSO (vehicle) and then subjected to a transmigration assay in response to CX3CL1 as described in Materials and Methods. n 3. , p 0.05 compared with CX3CL1-induced stimulation in vehicle- treated cells.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Migration, Lysis, Immunoprecipitation, Western Blot, Transmigration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 6. Cells with reduced Syk expression show impaired migration toward CX3CL1. A, Syk shRNA- treated cell migration in response to CX3CL1 was de- termined using a Transwell assay as described previ- ously and compared with NI and scr cells. n 4. , p 0.05 compared with CX3CL1-induced migration in NI cells. B, The ability of Syk shRNA and scr cells to mi- grate in response to 20 ng/ml CSF-1 was also evaluated. n 3.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Expressing, Migration, shRNA, Transwell Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 7. CX3CL1 activation of ERK1/2 is not required for RAW cell chemotaxis. A, RAW cells were stimulated with CX3CL1 for the indicated times before Western blotting of the corresponding Triton-soluble whole cell lysates. The activation statuses of ERK1/2 (p42/44MAPK), p38MAPK, and JNK1 were assessed using phosphospecific Abs, and the corresponding total (phosphorylation-independent) levels of MAPK expression are shown below as proof of equal protein loading. B, CX3CL1-induced ERK1/2 activation levels were compared between scrambled and Syk shRNA-treated cells. Results shown were obtained with a clone exhibiting 80% Syk protein reduction. C, RAW cells were preincubated for 1 h with 30 M PD98059 (MEK inhibitor) or with DMSO (vehicle) before being subjected to a transmigration assay. PD98059 efficacy was confirmed through its ability to abolish ERK1/2 phosphorylation (see inset). For all experiments, n 3.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Activation Assay, Chemotaxis Assay, Western Blot, Phospho-proteomics, Expressing, shRNA, Transmigration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 8. CX3CL1-induced cytoskeletal reorganization is disrupted in cells with reduced Syk expression. A, The ability of cells to increase their F-actin content after a 1-min CX3CL1 stimulation was compared among NI, scr, and Syk shRNA-treated (sh) cells, as previously described. n 4 independent experiments using heterogeneous cell populations. , p 0.05 compared with NI cells. B, Scrambled and Syk shRNA-treated cells were either untreated or treated with CX3CL1 for 1 min, and their ability to exhibit F-actin-rich cell protrusions (ruffles) was compared using F-actin staining. Representative images of three independent experiments are shown. Scale bar 10 m. The extent of CX3CL1-induced ruffles in individual cells was scored as described previously (18); protrusion in- dexes were calculated as the average of at least 50 cells in three different experiments and expressed as a percentage of scr cells. n 3. , p 0.05 compared with scrambled.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Expressing, shRNA, Staining