Fig 1: ORMDL3 overexpression impairs airway epithelium barrier integrity. ORMDL3 mRNA expression in (A) ORMDL3/Vector and (D) si-ORMDL3/si-control 16HBE cells showing successful overexpression and knockdown, respectively. After 24 h of culture, cells were collected, seeded in Transwell inserts and incubated for 72 h to form cell monolayers. (B) TEER of ORMDL3 in ORMDL3/Vector and (E) si-ORMDL3/si-control 16HBE monolayers were monitored at days 2 to 5. Epithelial permeability in (C) ORMDL3/Vector and (F) si-ORMDL3/si-control 16HBE cell monolayers were measured by treatment with 4-kDa FITC-dextran at day 5. Values are mean ± standard deviation (n=6 per group in duplicate). *P<0.05, **P<0.01 and ***P<0.001 vs. the corresponding control group. SPHK1, sphingosine kinase 1; ORMDL3, orosomucoid-like protein isoform 3; si, small interfering; Ctrl, control; TEER, transepithelial electrical resistance.
Fig 2: Expression levels of ORMDL3 protein in lung tissues of the (A) asthmatic model (B) budesonide-treated and (C) healthy control groups was investigated using immunohistochemistry. Magnification, ×100. ORMDL3 protein expression was quantified using (D) ImageJ analysis of stained sections and (E) western blotting. (F) mRNA expression level of ORMDL3 in lung tissues was determined using reverse transcription-quantitative polymerase chain reaction. (G) ORMDL3 protein expression was positively correlated with bronchial wall thickness, r=0.968, P<0.01. *P<0.05 and **P<0.01 vs. control; #P<0.05 and ##P<0.01 vs. treated. ORMDL3, orosomucoid-like 3.
Fig 3: OVA-RSV induces mouse airway inflammation and upregulates ORMDL3 expression. (A) Outline of the establishment of a chronic asthmatic mouse model with detailed strategy for OVA-RSV administration. i.p., i.n.h. and N.A. Histological analysis of lung tissues from control and OVA-RSV mice sacrificed on day 86. (B) Lung sections were stained with H&E to analyze the infiltration of inflammatory cells. (C) PAS staining was performed to assess goblet cell hyperplasia. (D) Masson's trichrome staining was carried out to evaluate sub-epithelial deposition of collagen and fibrosis. Values are mean ± standard deviation (n=6 per group). **P<0.01 vs. control group. Magnification, ×200. The results are representative of six independent animals. PAS, Periodic acid-Schiff; H&E, hematoxylin-eosin; i.n.h., inhalation; i.p., intraperitoneal; N.A., intranasal; OVA-RSV, ovalbumin-respiratory syncytial virus.
Fig 4: Expression levels of p-ERK protein in lung tissues of the (A) asthmatic model (B) budesonide-treated and (C) healthy control groups were investigated using immunohistochemistry. Magnification, ×100. (D) p-ERK protein expression was quantified using ImageJ analysis of stained sections and (E) western blotting. RNA expression level of MMP-9 in lung tissues was determined using reverse transcription-quantitative polymerase chain reaction. **P<0.01 vs. control; #P<0.05 vs. treated. (F) p-ERK protein expression was positively associated with bronchial wall thickness (r=0.813, P<0.01) and (G) ORMDL3 protein expression was positively associated with p-ERK level (r=0.727, P<0.01). (H) MMP-9 protein expression was positively associated with ORMDL3 expression level (r=0.914, P<0.01). p-ERK, phosphorylated-extracellular-signal regulated kinase; ORMDL3, orosomucoid-like 3; MMP-9, matrix metalloproteinase 9.
Fig 5: ORMDL3 overexpression downregulates TJ proteins. Assessment of Claudin-18 and E-cadherin mRNA expression levels in (A) ORMDL3/Vector and (B) si-ORMDL3/si-control 16HBE cells showing that ORMDL3 overexpression resulted in increased ORMDL3, reduced Claudin-18 and E-cadherin amounts, whereas ORMDL3 silencing led to reduced ORMDL3, elevated Claudin-18 and E-cadherin levels. The protein expression levels of (C) ORMDL3, (D) Claudin-18 and (E) E-cadherin in ORMDL3/Vector and the protein expression levels of (F) ORMDL3, (G) Claudin-18 and (H) E-cadherin in si-ORMDL3/si-control 16HBE cells were consistent with mRNA analysis. Immunofluorescence staining showing the localizations and signal intensities of Claudin-18 and E-cadherin in (I) ORMDL3/Vector and (J) si-ORMDL3/si-control 16HBE cells. Values are mean ± standard deviation (n=6 per group in duplicate). *P<0.05 and **P<0.01 vs. the corresponding control group. Arrows show the stratification of TJs. E, epithelial; SPHK1, sphingosine kinase 1; ORMDL3, orosomucoid-like protein isoform 3; si, small interfering; Ctrl, control; TNF, tumor necrosis factor; ERK, extracellular signal regulated kinase; TJ, tight junction.
Supplier Page from Abcam for Anti-ORMDL3 antibody