Journal: Cell Death & Disease

Article Title: Serpina3n attenuates granzyme B-mediated decorin cleavage and rupture in a murine model of aortic aneurysm

doi: 10.1038/cddis.2011.88

Figure Lengend Snippet: GZMB cleavage of decorin is prevented by pre-incubation with SA3N in vitro . Purified GZMB was pre-incubated with or without SA3N for 25 min at RT before incubation with recombinant human decorin for 24 h at RT. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane and visualized by western blotting for decorin ( a ) and Ponceau stain ( b ). In a , full-length decorin is indicated by an asterisk ( * ) and is visible at ∼65 kDa in lane 1. Decorin is severely reduced following incubation with GZMB in lane 2. In lane 3, decorin loss is not observed following pre-incubation of GZMB with SA3N. In b , SA3N and SA3N–GZMB complex at approximately 47 and 70 kDa, respectively (indicated by ^) are visible in lanes 2, 3 and 6. Full-length decorin is indicated by the asterisk ( * ) at approximately 65 kDa in lanes 4, 5 and 6. Decorin cleavage fragments in lane 5 are marked by arrows on the right side of the membrane

Article Snippet: The membrane was probed using a mouse anti-human decorin antibody (1 : 200, R&D Systems) and IRDye 800 conjugated affinity purified anti-mouse IgG (1 : 3000, Rockland Inc., Gilbertsville, PA, USA).

Techniques: Incubation, In Vitro, Purification, Recombinant, SDS Page, Membrane, Western Blot, Staining

Journal: Cancer cell international

Article Title: Revealing a novel Decorin-expressing tumor stromal subset in hepatocellular carcinoma via integrative analysis single-cell RNA sequencing.

doi: 10.1186/s12935-025-03811-0

Figure Lengend Snippet: Fig. 1 The expression and glycosylation of DCN in HCC and its correlation with patient survival. (A) The expression levels of DCN in the tumor (red) and peri-tumor (gray) tissues were revealed by GEPIA2. (B) The protein expression levels of DCN in normal liver and LIHC tumor tissues from The Human Protein Atlas. (C) Western blots of primary HCC (T) and peri-tumor (P) tissues with an anti-human DCN antibody. Red rectangle indicated chondroitin sulfate linked DCN (CS-DCN: 75 kDa to 150 kDa), and blue rectangle indicated DCN core protein (Core-DCN: 37 kDa). Actin was used as a loading control. (D) Quantification of DCN protein expression. Each signal was normalized to actin and shown as a relative intensity to 1P. The p values were calculated by paired T test. (E) Western blots of CS-DCN and core-DCN in HCC tissues. The cell lysate of HA59T was taken as an internal control of DCN intensity among experiments. Actin was used as a loading control. (F) Kaplan–Meier analysis of overall survival of HCC patients with high and low expressions of CS-DCN or core-DCN

Article Snippet: Total protein lysates (20–40 μg) of HCC tumor and peritumor tissue were separated by 8% SDS-PAGE and transferred to PVDF membrane, then blotted with antibodies against human DCN (R&D Systems, Minneapolis, MN, Catalog #: AF143) and Actin (Santa Cruz, sc-47778).

Techniques: Expressing, Glycoproteomics, Western Blot, Control

Journal: Cancer cell international

Article Title: Revealing a novel Decorin-expressing tumor stromal subset in hepatocellular carcinoma via integrative analysis single-cell RNA sequencing.

doi: 10.1186/s12935-025-03811-0

Figure Lengend Snippet: Fig. 4 Identification of the DCN-associated tumor stromal signature and its effect on HCC malignancy. (A) Venn diagram illustrating the origins of the CSPG and CS synthase genes in the signature. (B) Box plots showing the expression levels of the six signature genes across various cell types. *** P < 0.001 by one-way ANOVA. (C) Kaplan–Meier analyses for overall survival between high and low score groups of HCC patients from the TCGA-LIHC dataset (upper) and ICGC-LIRI-JP dataset (lower). The signature score for each sample was determined by ssGSEA based on the 6-gene DCN-associated signature. P value was calculated by log-rank test. (D) Quantification of recombinant DCN purification using UV gel electrophoresis. BSA was used as control. (E) Cell proliferation (CCK-8 assay) in HA22T cells and PLC5 cells with or without addition of recombinant DCN. Data represent the mean of three independent ex periments. Absorbance at 450 nm was measured and background-subtracted using medium-only controls (blank). (F) Transwell invasion assay of HA22T cells in matrigel with or without DCN. Data were expressed as the mean ± SD from three independent experiments

Article Snippet: Total protein lysates (20–40 μg) of HCC tumor and peritumor tissue were separated by 8% SDS-PAGE and transferred to PVDF membrane, then blotted with antibodies against human DCN (R&D Systems, Minneapolis, MN, Catalog #: AF143) and Actin (Santa Cruz, sc-47778).

Techniques: Expressing, Recombinant, Purification, Nucleic Acid Electrophoresis, Control, CCK-8 Assay, Transwell Invasion Assay

Journal: Autophagy

Article Title: DCN released from ferroptotic cells ignites AGER-dependent immune responses

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: DCN is a DAMP released during ferroptosis. (A) Cell death of HT1080 and HeLa cells following treatment with erastin (5 μM) for 24 h ( n = 3 biologically independent samples; * P < 0.05, one-tailed t test; data are presented as means ± SD). (B) An antibody array technology-based screening of protein release from HT1080 and HeLa cells following treatment with erastin (5 µM) for 24 h. The data are presented as the rank of fold change of protein release. (C) Top 10 released proteins in the setting of panel B. (D) ELISA assay of DCN release in HT1080 cells following treatment with erastin (5 μM), sulfasalazine (500 μM), sorafenib (10 μM), cysteine depletion, RSL3 (0.5 μM), ML162 (0.5 μM), ML210 (5 μM), FIN56 (2.5 μM), staurosporine (200 nM), or CCT137690 (5 μM) in the absence or presence of ferrostatin-1 (1 µM), Z-VAD-FMK (10 µM), or necrosulfonamide (1 μM) for 24 h (data are shown in a heat map as the means of three biologically independent samples). (E–G) HT1080 cells were treated with erastin (5 μM) or RSL3 (5 μM) in the absence or presence of glycine for 3–24 h and the release of DCN, HMGB1, and LDH were assayed by ELISA ( n = 3 biologically independent samples; * P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (H) Western blot assay of DCN in secreted proteins (SN) and whole cell lysates (WCL) in HT1080 cells following RSL3 (5 μM) treatment for 3 h.

Article Snippet: Commercially available ELISA kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), DCN (R&D Systems, DY143, DY1060), IL6 (R&D Systems, M6000B, D6050), TNF (R&D Systems, MTA00B, DTA00D), NFKB (Active Motif, 43,296), MPO (Abcam, ab105136), LDH (Abcam, ab102526), trypsin (Abcam, ab102531), CTSB (Abcam, ab119585), and AMY (Abcam, ab102523) in the indicated samples.

Techniques: One-tailed Test, Ab Array, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Autophagy

Article Title: DCN released from ferroptotic cells ignites AGER-dependent immune responses

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: DCN mediates inflammation caused by ferroptotic cells. (A) Ferroptotic cells lacking Dcn, Atg5, or Mcoln1 do not elicit the production of the pro-inflammatory TNF and IL6 cytokine by 1 × 10 6 BMDMs ( n = 3 biologically independent samples; * P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (B,C) Anti-DCN neutralizing antibody (1 mg/ml) inhibits ferroptotic cells, but not late apoptotic cell- or necroptotic cell-induced TNF and IL6 release in BMDMs ( n = 3 biologically independent samples; * P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (D,E) Lack of Dcn in late apoptotic or necroptotic cells fails to affect the production of TNF and IL6 by BMDMs ( n = 3 biologically independent samples; data are presented as means ± SD).

Article Snippet: Commercially available ELISA kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), DCN (R&D Systems, DY143, DY1060), IL6 (R&D Systems, M6000B, D6050), TNF (R&D Systems, MTA00B, DTA00D), NFKB (Active Motif, 43,296), MPO (Abcam, ab105136), LDH (Abcam, ab102526), trypsin (Abcam, ab102531), CTSB (Abcam, ab119585), and AMY (Abcam, ab102523) in the indicated samples.

Techniques:

Journal: Autophagy

Article Title: DCN released from ferroptotic cells ignites AGER-dependent immune responses

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: AGER mediates the response to DCN. (A) Western blot analysis of protein expression by BMDMs after transfection with the indicated shRNAs. (B) ELISA analysis of TNF and IL6 release in the indicated gene knockdown BMDMs following treatment with ferroptotic MEFs ( n = 3 biologically independent samples; * P < 0.05 versus control shRNA group, one-tailed t test; data are presented as means ± SD). (C) Lack of Ager in BMDMs blocks ferroptotic cell-induced the production of the pro-inflammatory TNF and IL6 cytokines ( n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (D) Anti-AGER neutralizing antibody (Ab; 1 mg/ml), but not anti-TLR4 neutralizing antibody (1 mg/ml), inhibits ferroptotic cell-induced TNF and IL6 release in BMDMs ( n = 3 biologically independent samples; * P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (E, F) Lack of Ager in BMDMs inhibits DCN-induced TNF and IL6 release in the absence or presence of HMGB1 ( n = 3 biologically independent samples; * P < 0.05 versus WT group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (G, H) Analysis of NFKB activity and TNF release in the indicated BMDMs following treatment with ferroptotic cells or DCN in the absence or presence of the NFKB pathway inhibitor BMS-345541 ( n = 3 biologically independent samples; * P < 0.05 versus WT group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (I) His-tag affinity pull-down analysis of the binding of DCN to AGER.

Article Snippet: Commercially available ELISA kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), DCN (R&D Systems, DY143, DY1060), IL6 (R&D Systems, M6000B, D6050), TNF (R&D Systems, MTA00B, DTA00D), NFKB (Active Motif, 43,296), MPO (Abcam, ab105136), LDH (Abcam, ab102526), trypsin (Abcam, ab102531), CTSB (Abcam, ab119585), and AMY (Abcam, ab102523) in the indicated samples.

Techniques: Western Blot, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Knockdown, Control, shRNA, One-tailed Test, Activity Assay, Binding Assay

Journal: Autophagy

Article Title: DCN released from ferroptotic cells ignites AGER-dependent immune responses

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: Inhibiting the DCN-AGER pathway protects against cerulein-induced acute pancreatitis. WT and pancreatic gpx4 −/− mice received anti-DCN Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 161026), anti-AGER Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 175410), control IgG (20 mg/kg; R&D Systems) or liproxsatin-1 (10 mg/kg) by i.p. injection 2 h after completion of the cerulein-induced pancreatitis protocol. (A) Pancreatic sections were stained with H&E at 16 h (bar: 100 μm). (B–G) Serum AMY (amylase), pancreatic trypsin activity, pancreatic MPO activity, serum DCN, serum TNF, and serum IL6 were assayed at 8 and 16 h ( n = 5 mice/group; * P < 0.05 versus IgG group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD).

Article Snippet: Commercially available ELISA kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), DCN (R&D Systems, DY143, DY1060), IL6 (R&D Systems, M6000B, D6050), TNF (R&D Systems, MTA00B, DTA00D), NFKB (Active Motif, 43,296), MPO (Abcam, ab105136), LDH (Abcam, ab102526), trypsin (Abcam, ab102531), CTSB (Abcam, ab119585), and AMY (Abcam, ab102523) in the indicated samples.

Techniques: Control, Injection, Staining, Activity Assay

Journal: Journal of Thrombosis and Haemostasis

Article Title: Lytic and mechanical stability of clots composed of fibrin and blood vessel wall components

doi: 10.1111/jth.12112

Figure Lengend Snippet: Fibrin clot lysis assay. Fibrin clots containing plasmin and the indicated additives at final concentrations 50 μg mL −1 for collagen fragments, dermatan sulfate or chondroitin sulfate and 50 n m for decorin core protein or aorta decorin were prepared and the absorbance was continuously measured at 340 nm (turbidity, A 340 ). Mean values of eight measurements from three independent experiments (continuous lines) and standard deviation (SD) values above and below mean (dotted lines) are shown. Symbols indicate half-maximal values of A 340 reported as lysis time in .

Article Snippet: Recombinant human decorin core protein was a product of R&D Systems (Abingdon, England).

Techniques: Lysis, Standard Deviation

Journal: Journal of Thrombosis and Haemostasis

Article Title: Lytic and mechanical stability of clots composed of fibrin and blood vessel wall components

doi: 10.1111/jth.12112

Figure Lengend Snippet: Penetration of tissue plasminogen activator (tPA)-YFP into fibrin in the course of lysis. Clots were prepared from fibrinogen containing Alexa 546 -labeled fibrinogen, plasminogen and the indicated additives at final concentrations 50 μg mL −1 for collagen fragments, dermatan sulfate or chondroitin sulfate and 50 n m for decorin core protein or aorta decorin. After 30-min clotting tPA-YFP was added to fibrin and the fluid/fibrin interface was monitored by confocal laser scanning microscopy using double fluorescent tracing (excitation 488 nm/emission 525 nm for tPA-YFP and excitation 543 nm/emission 575 nm for fibrin). Images are shown for the 25th min after the application of tPA-YFP, scale bar = 100 μm. The tPA-related fluorescence stains in green, whereas the fibrin is shown in red in these images and selected regions are digitally magnified four-fold to illustrate the structural pattern of the interface layer. At 0 time the edge of the fibrin clot was approximately at the same position near the top of each field of observation. The numbers in each panel indicate the distance for penetration of tPA-YFP in the clot at 25 min (mean and standard deviation from three samples, P < 0.05 for all additives according to the Kolmogorov–Smirnov test in comparison to pure fibrin).

Article Snippet: Recombinant human decorin core protein was a product of R&D Systems (Abingdon, England).

Techniques: Lysis, Labeling, Coagulation, Confocal Laser Scanning Microscopy, Fluorescence, Standard Deviation, Comparison

Journal: Journal of Thrombosis and Haemostasis

Article Title: Lytic and mechanical stability of clots composed of fibrin and blood vessel wall components

doi: 10.1111/jth.12112

Figure Lengend Snippet: Ultrastructure of the fibrin network. (A) Fibrinogen (4.8 μ m ) without any additive (i) or containing 50 n m fully glycosylated decorin; and (ii) was mixed with thrombin and after 1 h clots were fixed with glutaraldehyde, dehydrated and examined with scanning electron microscopy (SEM). Scale bar = 1 μm. (B) Morphometric analysis of fibrin structure. Fibrin clots containing the indicated additives were prepared and SEM images taken as illustrated in panel A. The diameter of 300 fibers was measured and their empiric (black histograms) as well as best-fitted theoretical (gray curves) probability density function (PDF) was determined. Median values and the interquartile range are shown for the theoretical distributions of the diameter values.

Article Snippet: Recombinant human decorin core protein was a product of R&D Systems (Abingdon, England).

Techniques: Electron Microscopy

Journal: Journal of Thrombosis and Haemostasis

Article Title: Lytic and mechanical stability of clots composed of fibrin and blood vessel wall components

doi: 10.1111/jth.12112

Figure Lengend Snippet: Modification of the viscoelastic properties of fibrin by extracellular matrix components. (A) Fibrinogen (7.4 μ m ) containing 50 n m decorin (fully glycosylated aorta or core protein) was mixed with thrombin and the storage modulus (G', continuous line) and the loss modulus (G'', crossed line) were measured using an oscillation rheometer. (B) After 15-min clotting, stepwise increasing shear stress τ was applied to fibrin formed in the gap space of the oscillation rheometer and viscosity ( η ) was measured. The critical values τ 0 , which define the gel/fluid transition of fibrin, are shown in .

Article Snippet: Recombinant human decorin core protein was a product of R&D Systems (Abingdon, England).

Techniques: Modification, Coagulation, Shear, Viscosity

Journal: Journal of Thrombosis and Haemostasis

Article Title: Lytic and mechanical stability of clots composed of fibrin and blood vessel wall components

doi: 10.1111/jth.12112

Figure Lengend Snippet: Plasma clot lysis. Citrated human plasma was supplemented with 50 ng mL −1 tissue plasminogen activator (tPA) and the indicated additives at final concentrations 50 μg mL −1 for collagen fragments, dermatan sulfate or chondroitin sulfate and 50 n m for decorin core protein or aorta decorin. Clotting was initiated with CaCl 2 and thrombin at 12.5 m m and 15 n m final concentrations, respectively, and the absorbance was continuously measured at 340 nm (turbidity, A 340 ). Mean values of eight measurements from three independent experiments (continuous lines) and standard deviation (SD) values above and below the mean (dotted lines) are shown. Symbols indicate half-maximal values of A 340 , P < 0.05 for all additives according to the Kolmogorov–Smirnov test in comparison to additive-free clot lysis time.

Article Snippet: Recombinant human decorin core protein was a product of R&D Systems (Abingdon, England).

Techniques: Clinical Proteomics, Lysis, Coagulation, Standard Deviation, Comparison

Journal: Journal of Sport and Health Science

Article Title: Decorin, an exercise-induced secretory protein, is associated with improved prognosis in breast cancer patients but does not mediate anti-tumorigenic tissue crosstalk in mice

doi: 10.1016/j.jshs.2024.100991

Figure Lengend Snippet: Systemic delivery of recombinant decorin into SCID mice injected with MDA-MB-231-HM cells. SCID mice injected with the MDA-MB-231-HM cells into mammary pads received 2 different doses of recombinant decorin systemically. (A) Tumor volume, (B) metastatic burden, (C) plasma decorin levels, and (D) tumor decorin levels after the low-dose experiment. Mice were dosed every second day with 100 µg of recombinant decorin or an equal volume of saline (Control) ∼14 days post tumor-cell inoculation. (E) Tumor volume, (F) metastatic burden, (G) plasma decorin levels, and (H) tumor decorin levels after the high-dose experiment. Mice were dosed daily with 10 mg/kg body weight (∼2.5 times the dose of low-dose experiment) of recombinant decorin or an equal volume of saline (Control) ∼7 days post tumor-cell inoculation. Tumor volume was assessed by calliper measurement of primary tumor growth and calculated as (length × width )/2. Metastatic burden was assessed by bioluminescence imaging detection. Plasma decorin levels were measured by ELISA. Tumor decorin levels were measured by western blot using total protein normalization. Two AAV:Dcn gastrocnemius lysates from the overexpression experiments were used as positive controls. Statistical testing was done with two-way analyses of variance or mixed effects model for repeated measures or Student's t test. Data expressed as means ± SEM. ( n = 5–7 mice per group). A.U. = arbitrary unit; con = control; Dcn = decorin; ELISA = enzyme linked immunosorbent assay.

Article Snippet: The concentration of decorin was measured in EV-free plasma with enzyme linked immunosorbent assay (ELISA) (DuoSet, Cat# DY143; R&D Systems, Minneapolis, MN, USA).

Techniques: Recombinant, Injection, Clinical Proteomics, Saline, Control, Imaging, Enzyme-linked Immunosorbent Assay, Western Blot, Over Expression