sdc4  (R&D Systems)

Journal: Scientific Reports

Article Title: Glycoprotein non-metastatic melanoma protein B is a potential biomarker for arthroplasty aseptic loosening

doi: 10.1038/s41598-025-13922-3

Figure Lengend Snippet: Transcriptome analysis of eight sex- and age-balanced BM-MSC preparations by RNA-seq. ( A ) Volcano plot emphasizing differentially expressed genes with DESeq2-based p adj -value cut-off 0.05. These genes were included in the gene set enrichment analysis in C , D . Arrows highlight GPNMB and SDC4 . ( B ) Heatmap showing differentially expressed genes with DESeq2-based p adj -value cut-off 0.05. Qlucore Omics Explorer was used for the heatmap visualisation and normalization of the data (each column has mean of 0 and variance of 1) of four primary hip surgeries vs. four revisions (2x hip, 2x knee). Arrows highlight GPNMB and SDC4 . ( C ) KEGG analyses (KEGG: https://www.kegg.jp/ ; of downregulated genes (upper panel) as identified using DESeq2. Gene ontologies for Biological Processes (middle panel) and Molecular Functions (lower panel) are included. ( D ) KEGG analyses of upregulated genes (upper panel) as identified using DESeq2. Gene ontologies for Biological Processes (middle panel) and Molecular Functions (lower panel) are included.

Article Snippet: Afterwards, sandwich ELISA was performed to analyse the secretion of GPNMB (Human Osteoactivin/GPNMB DuoSet ELISA; DY2550, R&D Systems) or SDC4 (Human Syndecan-4 DuoSet ELISA; DY2918, R&D Systems) according to the manufacturer’s protocols.

Techniques: RNA Sequencing

Journal: Scientific Reports

Article Title: Glycoprotein non-metastatic melanoma protein B is a potential biomarker for arthroplasty aseptic loosening

doi: 10.1038/s41598-025-13922-3

Figure Lengend Snippet: Transcriptome analysis of all primary vs. all revision arthroplasties at both implantation sites (28 samples). ( A ) KEGG analyses (KEGG: https://www.kegg.jp/ ; of downregulated genes (upper panel). Gene ontologies for Biological Processes (middle panel) and Molecular Functions (lower panel) are included. ( B ) KEGG analyses of upregulated genes (upper panel). Gene ontologies for Biological Processes (middle panel) and Molecular Functions (lower panel) are included. ( C ) RNA-seq data for GPNMB in BM-MSCs (normalised counts). For the statistical comparisons between primary and revision the parametric unpaired Welch’s t-test was used. Experimental data are shown as individual values. ( D , E ) GPNMB relative expression levels in BM-MSCs based on RPS29 as housekeeping gene. E indicates the levels for individual samples. ( F ) GPNMB relative expression levels in MNCs from blood (PB) and synovial fluid (SF). ( G ) RNA-seq data for SDC4 in BM-MSCs (normalised counts). For the statistical comparisons between primary and revision the parametric unpaired Welch’s t-test was used. Experimental data are shown as individual values. (H , I) SDC4 relative expression levels in BM-MSCs based on RPS29 as housekeeping gene. I indicates the levels for individual samples. ( J ) SDC4 relative expression levels in MNCs from blood (PB) and synovial fluid (SF). The black bar in C, D, G, H represents the median value. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Afterwards, sandwich ELISA was performed to analyse the secretion of GPNMB (Human Osteoactivin/GPNMB DuoSet ELISA; DY2550, R&D Systems) or SDC4 (Human Syndecan-4 DuoSet ELISA; DY2918, R&D Systems) according to the manufacturer’s protocols.

Techniques: RNA Sequencing, Expressing

Journal: Diabetes

Article Title: Adiponectin reduces glomerular endothelial glycocalyx disruption and restores glomerular barrier function in a mouse model of type 2 diabetes.

doi: 10.2337/db23-0455

Figure Lengend Snippet: Figure 3—Adiponectin ameliorates TNFa-induced glycocalyx shedding. GEnCs were treated with or without 10 ng/mL TNF-a for 2 h in the presence or absence of 2.5 mg/mL gAd. A) qPCR analysis of SDC4 mRNA levels, normalized to GAPDH, are shown. Data are plotted as the mean 2-(DCT) (gene of interest/housekeeping gene) of each triplicate with the mean. B) SDC4 protein expression was quantified by ELISA in the medium of treated cells. C) Sulfated GAGs were quantified in the medium of treated cells by Alcian blue colorimetric assay. MMP2 (D) and MMP9 (E) mRNA levels in CiGEnCs treated with TNF-a (10 ng/mL) and/or gAd (2.5 mg/mL). A–E) One-way ANOVA (n = 5); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by post hoc analysis (Bonferroni). MMP2 was knocked down using three shRNA dif- ferent sequences in GEnCs (v33, v47, v49) or not, using scrambled controls. F) qPCR data analysis highlighting the decreased expression of MMP2 mRNA in CiGEnCs by all three different shRNA sequences. Data are plotted as the mean comparative cycle threshold of each triplicate. GAPDH used as the housekeeping gene control, n = 4; one-way ANOVA, ****P < 0.0001 post hoc analysis (Bonferroni). Gi) Representative Western Blot demonstrating the protein knockdown of MMP2 by shRNA clone v47. Gii) Densitometry showed significant knockdown of MMP2 protein expression by clone v47. Data are normalized to the b-actin loading control. Dots represent mean ± SEM; one-way ANOVA, ***P < 0.001 by post hoc analysis (Bonferroni). H) SDC4 mRNA levels in MMP2 knockdown CiGEnCs treated with TNF-a and/or gAd. Data are plotted as the mean comparative cycle threshold of each triplicate. GAPDH was used as the housekeeping gene control, n = 5; one-way ANOVA, ***P < 0.001 post hoc analysis (Bonferroni).

Article Snippet: Cellular levels of SDC4 were quantified using a SDC4 ELISA (R&D Systems; catalog DY2918) per the manufacturer’s instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, shRNA, Control, Western Blot, Knockdown

Journal: Diabetes

Article Title: Adiponectin reduces glomerular endothelial glycocalyx disruption and restores glomerular barrier function in a mouse model of type 2 diabetes.

doi: 10.2337/db23-0455

Figure Lengend Snippet: Figure 4—Adiponectin signals to glomeruli directly and prevents the mRNA upregulation of glycocalyx-related genes in db/db glomeruli. Hu- man (A) and mouse (B) glomeruli were isolated and treated ex vivo with 2.5 mg/mL gAd. Representative Western Blots demonstrating gAd ef- fects at 30 min in human (Ai) and mouse (Bi) ex vivo glomeruli on AMPK phosphorylation (p-). Aii and Bii) Densitometry confirmed significant phosphorylation of AMPK in response to gAd after 30 min in human and mouse glomeruli. Densitometry was performed on blots from inde- pendent repeats (n = 4) showing levels of protein of interest normalized to the b-actin loading control. Dots represent mean ± SEM; one-way ANOVA; *P < 0.05, by post hoc analysis (Bonferroni). C–F) qPCR analysis graph representing the mRNA expression of TNF (C), SDC4 (D), MMP2 (E), and MMP9 (F) in ex vivo (wild type or db/db) sieved glomeruli treated with or without adiponectin. Data are plotted as the mean comparative cycle threshold of each triplicate, n = 5; one-way ANOVA; *P < 0.05, **P < 0.01, ****P < 0.0001 by post hoc analysis (Bonferroni).

Article Snippet: Cellular levels of SDC4 were quantified using a SDC4 ELISA (R&D Systems; catalog DY2918) per the manufacturer’s instructions.

Techniques: Isolation, Ex Vivo, Western Blot, Phospho-proteomics, Control, Expressing

Journal: Kidney International

Article Title: Blocking matrix metalloproteinase-mediated syndecan-4 shedding restores the endothelial glycocalyx and glomerular filtration barrier function in early diabetic kidney disease

doi: 10.1016/j.kint.2019.09.035

Figure Lengend Snippet: Syndecan-4 ( S dc 4 ) mRNA and protein are altered in early diabetic kidney disease (DKD). Glomerular endothelial cells (GECs) were sorted by fluorescence-activated cell sorting, and ( a ) S dc 4 and ( b ) S dc 1 mRNA expression were determined. The 2 −ΔΔCT method of quantification was used to calculate the fold change, normalized to glyceraldehyde-3-phosphate dehydrogenase, at week 8 post-streptozotocin (STZ), ( S dc 4 : control, 1.000 ± 0.2565, n = 9 mice; diabetes, 2.470 ± 0.6580, n = 8 mice; * P < 0.05; S dc 1 : control, 1.000 ± 0.1112, n = 5 mice; diabetes, 1.663 ± 0.4289, n = 5 mice; nonsignificant [NS]). Control and diabetic kidney sections were labeled with SDC4 and endothelial membrane label R18. ( c ) Peak-to-peak assessment of the glomerular endothelial glycocalyx using SDC4 labeling at week 9 post-STZ showed a significant reduction in glycocalyx SDC4 in diabetes (control, 182.5 ± 12.04, n = 5 mice; diabetes, 64.31 ± 6.513, n = 5 mice; *** P < 0.0001). Isolated glomerular lysates from control and diabetic mice were used to determine ( d ) SDC4 and ( e ) SDC1 concentration using SDC4 and SDC1 ectodomain enzyme-linked immunosorbent assay (ELISA). The data were then normalized to total protein content. SDC4 (control, 1.327 ± 0.1439, n = 6 mice; diabetes, 0.8059 ± 0.1218, n = 6 mice; * P < 0.05); SDC1 (control, 9.671 ± 1.742, n = 6 mice; diabetes, 9.699 ± 1.313, n = 6 mice; NS) at week 8 post-STZ. SDC4 shedding in the ( f ) plasma and ( g ) urine was determined in DKD using the SDC4 ectodomain ELISA at week 8 post-STZ. For plasma: control, 2.631 ± 0.2860, n = 6 mice; diabetes, 4.801 ± 0.3793, n = 6 mice; ** P < 0.005. Urine SDC4 was normalized to creatinine (control, 3.343 ± 0.9410, n = 4 mice; diabetes, 39.32 ± 13.03, n = 4 mice; * P < 0.05; Mann-Whitney test at week 8 post-STZ). Each dot or square on the graph represents a mouse. Data are expressed as the mean ± SEM, and unpaired Student t test was used for statistical analysis unless specified.

Article Snippet: The concentrations of SDC4 (ectodomain) in mouse glomerular lysate, plasma, and urine were quantified using a sandwich enzyme immunoassay (mouse SDC4 ELISA kit, CSB-EL020891MO; Cusabio, Houston, TX) according to the manufacturer’s instructions.

Techniques: Fluorescence, FACS, Expressing, Control, Labeling, Membrane, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, MANN-WHITNEY

Journal: Kidney International

Article Title: Blocking matrix metalloproteinase-mediated syndecan-4 shedding restores the endothelial glycocalyx and glomerular filtration barrier function in early diabetic kidney disease

doi: 10.1016/j.kint.2019.09.035

Figure Lengend Snippet: Blockade of matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) ameliorates diabetes-induced changes in syndecan-4 (SDC4) and reduces plasma MMP activity. Diabetic (Dia) + MMP2 and MMP9 inhibitor (MMPI) or vehicle (Veh)-treated kidney sections were labeled with SDC4 and endothelial membrane label R18. ( a ) Peak-to-peak assessment of the glomerular endothelial glycocalyx (eGLX) using SDC4 labeling at week 9 post-streptozotocin showed a significant restoration in glycocalyx thickness (Dia Veh, 109.2 ± 9.003, n = 5 mice; Dia MMPI, 246.0 ± 22.36, n = 5 mice; *** P = 0.0005). ( b ) Isolated glomerular lysate from diabetic + MMPI or vehicle-treated mice was used to determine S dc 4 mRNA expression. The 2 −ΔΔCT method of quantification was used to calculate the fold change, normalized to glyceraldehyde-3-phosphate dehydrogenase and relative to Dia Veh (Dia Veh, 1.000 ± 0.089, n = 11 mice; Dia MMPI, 0.6658 ± 0.04849, n = 12 mice; ** P = 0.0029). ( c ) Isolated glomerular lysate from diabetic + MMPI or vehicle-treated mice was used to determine SDC4 concentration with previously used SDC4 ectodomain enzyme-linked immunosorbent assay. The data were then normalized to total protein content (Dia Veh, 0.6426 ± 0.06780, n = 5 mice; Dia MMPI, 1.150 ± 0.1634, n = 7 mice; * P = 0.0322). MMPI attenuated SDC4 shedding in the ( d ) plasma (Dia Veh, 1.000 ± 0.1029, n = 11 mice; Dia MMPI, 0.5449 ± 0.09749, n = 9 mice; ** P = 0.0054), but not in the ( e ) urine (Dia Veh, 1.000 ± 0.1895, n = 13 mice; Dia MMPI, 0.8501 ± 0.1290, n = 8 mice; nonsignificant [NS]) in diabetic kidney disease. The fold change of diabetic MMPI relative to diabetic vehicle was calculated to enable pooling of results from different experiments. ( f,g ) Plasma MMP2 (Dia Veh, 3.001 ± 0.3977, n = 13 mice; Dia MMPI, 1.528 ± 0.4924, n = 7 mice; * P = 0.0366) and MMP9 (Dia Veh, 1.150 ± 0.07157, n = 9 mice; Dia MMPI, 0.9266 ± 0.03281, n = 6 mice; * P = 0.0314) activities, using MMP2 and MMP9 Biotrak Activity Assays (GE Healthcare Life Sciences, Buckinghamshire, UK), were reduced in the diabetic + MMPI group when compared with the diabetic + vehicle group. Each dot or square on the graph represents a mouse. Data are expressed as the mean ± SEM, and unpaired Student t test at week 9 post-streptozotocin was used for statistical analysis unless specified.

Article Snippet: The concentrations of SDC4 (ectodomain) in mouse glomerular lysate, plasma, and urine were quantified using a sandwich enzyme immunoassay (mouse SDC4 ELISA kit, CSB-EL020891MO; Cusabio, Houston, TX) according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Activity Assay, Labeling, Membrane, Isolation, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay