human decorin duoset elisa  (R&D Systems)

Journal: Biochemistry and Biophysics Reports

Article Title: Eggshell membrane and its major component lysozyme and ovotransferrin enhance the secretion of decorin as an endogenous antifibrotic mediator from lung fibroblasts and ameliorate bleomycin-induced pulmonary fibrosis

doi: 10.1016/j.bbrep.2024.101806

Figure Lengend Snippet: (A–C) Eggshell membrane and its components LYZ and LYZ + TF increase DCN secretion from fibroblasts. Quantifying secreted DCN from human lung fibroblasts WI-38 with or without TGF-β (5 ng/mL) stimulation upon addition of ESM (A), LYZ (B), and LYZ + TF (C) by ELISA (n = 3, *p < 0.05, **p < 0.01). The LYZ in the ESM was about 50 % based on proteomic analysis. The protein content in the hydrolyzed ESM used in (A) was 20 %. Thus, the minimum amount of LYZ added was set at approximately 10 % of 1 mg/ml (A). (D–I) Eggshell membrane treatment inhibited the TGF-β-induced fibrosis pathway in WI-38. Immunofluorescence staining for TAZ with or without TGF-β (5 ng/mL) stimulated WI-38 human lung fibroblasts with or without 1 mg/mL ESM in the medium. E. Quantifying nuclear/cytoplasmic ratios of TAZ staining in WI-38 with or without TGF-β (5 ng/mL) stimulation and with or without ESM (ESM (−): 0 mg/mL and ESM (+): 1 mg/mL). F. Suppressing DCN secretion by dcn siRNA to WI-38. N = 3. G. dcn knockdown by siRNA causes nuclear localization of TAZ without TGF-β stimulation (n = 15; 3 dish × 5 observations). H. Eggshell membrane treatment inhibited the TGF-β-induced fibrosis pathway in human lung fibroblasts. Immunofluorescence staining for pSmad2 with or without TGF-β (5 ng/mL) stimulated WI-38 human lung fibroblasts stimulated with or without 1 mg/mL ESM in the medium. I. Quantifying nuclear/cytoplasmic ratios of pSmad2 staining in WI-38 stimulated with or without TGF-β (5 ng/mL) in the presence or absence of ESM (ESM (−): 0 mg/mL and ESM (+): 1 mg/mL). DCN, Decorin; ESM, eggshell membrane; LYZ; lysozyme; TF, Ovotransferrin; TGF-β, Transforming growth factor-β.

Article Snippet: Cells were treated with 5 ng/mL recombinant human TGF-β1 (Abcam, Cambridge, UK) for 5 h and culture supernatants were collected before fixing the cells for DCN quantification using Decorin DuoSet enzyme linked immunosorbent assay (ELISA) (#DY143; R&D Systems, Minneapolis, MN, USA).

Techniques: Membrane, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Knockdown

Journal: Frontiers in Pharmacology

Article Title: Substrate stiffness engineered to replicate disease conditions influence senescence and fibrotic responses in primary lung fibroblasts

doi: 10.3389/fphar.2022.989169

Figure Lengend Snippet: Primers and probes with designated exon boundaries.

Article Snippet: Secreted IL-6 (DY205), CXCL8 (DY208), DCN (DY143), TGF-β1 (DY240), Osteoprotegerin (OPG; DY805), receptor activator of nuclear factor kappa-B ligand (RANKL; DY626) and Connective Tissue Growth Factor (CTGF) (DY9190-05) were measured using Human DuoSet Enzyme linked immunosorbent assay (ELISA) kits (R&D Systems, Abingdon, United Kingdom) according to the manufacturer’s instructions.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Substrate stiffness engineered to replicate disease conditions influence senescence and fibrotic responses in primary lung fibroblasts

doi: 10.3389/fphar.2022.989169

Figure Lengend Snippet: Stiffness induced a secretory phenotype in Primary human lung fibroblasts cultured on stiff matrices. (A,B) DCN gene expression and secreted protein were measured after 7 days of culture on soft (3.94 ± 0.87 kPa) and stiff (11.34 ± 3.82 kPa) hydrogels. (C–H) Levels of secreted factors RANKL, OPG, CTGF and TGF-β1 including TGF-β1 and CTGF gene expression after 7 days of culture on soft and stiff hydrogels. I) Correlation was calculated between OPG, RANKL and all other secreted factors. Panel J shows a fluorescent staining for DCN, DAPI was used for identification of cell nuclei. Illustrated photographs are representative images of in total 7 unique donors. Gene expression data were normalised against 18S and expressed as 2 −ΔCT x 10 6 . Secreted matrix proteins and cytokines were normalised to cell counts and expressed as pg/ml or ng/ml 10 5 cells. Differences between soft and stiff hydrogels were analysed by a Wilcoxon matched pairs signed rank test. Correlation was calculated using Pearson correlation coefficient. ( n = 6) Scale bar = 50 µm. Each donor has a different unique colour (blue, red, yellow, green, black, and grey).

Article Snippet: Secreted IL-6 (DY205), CXCL8 (DY208), DCN (DY143), TGF-β1 (DY240), Osteoprotegerin (OPG; DY805), receptor activator of nuclear factor kappa-B ligand (RANKL; DY626) and Connective Tissue Growth Factor (CTGF) (DY9190-05) were measured using Human DuoSet Enzyme linked immunosorbent assay (ELISA) kits (R&D Systems, Abingdon, United Kingdom) according to the manufacturer’s instructions.

Techniques: Cell Culture, Gene Expression, Staining