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