Journal: Biochemical Journal

Article Title: Heparan sulfate 6-O-endosulfatases: discrete in vivo activities and functional co-operativity

doi: 10.1042/bj20060848

Figure Lengend Snippet: Figure 6 Loss of mSulf activity increases the mitogenic response of MEFs to FGF2

Article Snippet: Recombinant human FGF2 and HGF/SF (HGF/scatter factor) were from R&D Systems (Abingdon, Oxon., U.K.).

Techniques: Activity Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

doi: 10.1161/ATVBAHA.120.315398

Figure Lengend Snippet: Figure 3. Gal-1 (galectin-1) is induced by TNFα (tumor necrosis factor alpha) and enhances TNFα-induced MMP (matrix metalloprotease)-9 expression. A, Cultured vascular smooth muscle cells (VSMCs) and adventitial fibroblasts were treated without (control [ctrl]) or with Ang II (angiotensin II; 100 nmol/L), TNFα (100 ng/mL), or IL (interleukin)-1β (10 ng/mL) as indicated for 48 h. Gal-1 expression in cell lysates was examined by Western blot analysis. B, The Gal-1 levels in culture media collected from treated cells described in A were measured by ELISA. Data shown are mean±SE of 3 independent experiments. *P<0.05 vs ctrl. C, Adventitial fibroblasts isolated from WT (wild type) and Gal-1−/− mice were treated with indicated concentrations of TNFα in culture for 48 h. Culture media were then collected and analyzed by gelatin zymography. Cell lysates were prepared and subjected to Western blot analysis using indicated antibodies. D, WT and Gal-1−/− fibroblasts were treated with TNFα (100 ng/mL) for indicated times. Cell lysates were prepared, and the expression levels of phosphorylated Erk1/2 and total Erk1/2 were examined by Western blot analysis. E, WT and Gal-1−/− fibroblasts were treated with or without TNFα (100 ng/mL) in the absence or presence of PD98095 (10 µmol/L) as indicated in culture for 48 h. Culture media and cell lysates were subjected to zymography and Western blot analysis, respectively.

Article Snippet: After collection and 3 washes, cells were resuspended in DMEM containing 10% FBS and 1 ng/mL of basic fibroblast growth factor (3139-FB; R&D Systems), followed by plating on 6-cm dish.

Techniques: Expressing, Cell Culture, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Zymography

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

doi: 10.1161/ATVBAHA.120.315398

Figure Lengend Snippet: Figure 4. Gal-1 (galectin-1) oxidation promotes MMP (matrix metalloprotease)-9 and inflammatory cytokine expression. A, Tissue lysates prepared from freshly isolated vessels of apoE-deficient mice were incubated with 2 mmol/L biotin-polyethylene oxide (PEO) iodoacetamide preceded with or without tris(2-carboxyethyl) phosphine hydrochloride (TCEP; 5 mmol/L) treatment at room temperature for 2 h in dark. The lysates were then subjected to immunoprecipitation with control IgG or anti-Gal-1 antibody as indicated. The levels of biotinylation and Gal-1 in cell lysates and immunoprecipitates were examined by streptavidin blotting and Western blot analysis, respectively. B, Cultured macrophages, vascular smooth muscle cells (VSMCs), and adventitial fibroblasts from Gal-1−/− mice were incubated with indicated concentrations of CSGal-1 (cysteine-less Gal-1 mutant) or oxidized Gal-1 (oxGal-1) for 24 (macrophages) or 48 h (VSMCs and fibroblasts). Culture media were harvested and analyzed by zymography. Cell lysates were prepared and subjected to Western blot analysis with MMP9 and β-actin antibodies. C, The levels of TNFα (tumor necrosis factor alpha), IL (interleukin)-6, and MCP-1 (monocyte chemoattractant protein-1) in culture media harvested from treated cells described in B were determined by ELISA. Data shown are mean±SE of 4 independent experiments. *P<0.05; **P<0.01, ***P<0.001 vs control.

Article Snippet: After collection and 3 washes, cells were resuspended in DMEM containing 10% FBS and 1 ng/mL of basic fibroblast growth factor (3139-FB; R&D Systems), followed by plating on 6-cm dish.

Techniques: Expressing, Isolation, Incubation, Immunoprecipitation, Control, Western Blot, Cell Culture, Mutagenesis, Zymography, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

doi: 10.1161/ATVBAHA.120.315398

Figure Lengend Snippet: Figure 5. MAP kinase pathways are implicated in oxidized Gal-1 (galectin-1; oxGal-1)–mediated induction of MMP (matrix metalloprotease)-9 and inflammatory cytokines. A, Cultured macrophages, vascular smooth muscle cells (VSMCs), and fibroblasts were treated with 1 µg/mL of oxGal-1 in culture for indicated times. The phosphorylation states of Erk, JNK, and p38 kinases were examined by Western blot analysis. B, Cultured macrophages, VSMCs, and fibroblasts were incubated without or with 1 µg/mL of oxGal-1 in the absence or presence of 10 µmol/L Erk inhibitor (PD98059), 10 µmol/L JNK inhibitor (SP600125), or 20 µmol/L p38 inhibitor (SB203580) as indicated for 24 (macrophages) or 48 h (VSMCs and fibroblasts). The cultured media were harvested, and the levels of indicated cytokines were determined by ELISA. Data shown are mean±SE of 3 to 4 independent experiments. Ctrl indicates control; IL-6, interleukin; MCP-1, monocyte chemoattractant protein-1; and TNFα, tumor necrosis factor alpha. *P<0.05; **P<0.01 vs oxGal-1–treated group without coincubation with respective kinase inhibitor.

Article Snippet: After collection and 3 washes, cells were resuspended in DMEM containing 10% FBS and 1 ng/mL of basic fibroblast growth factor (3139-FB; R&D Systems), followed by plating on 6-cm dish.

Techniques: Cell Culture, Phospho-proteomics, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal:

Article Title: Serosal Mesothelium Retains Vasculogenic Potential

doi: 10.1002/dvdy.21334

Figure Lengend Snippet: Growth factors induce epithelial–mesenchymal transition (EMT) and smooth muscle actin (SMA) expression in serosal mesothelial cultures. Explants were cultured for 5 days under various conditions and analyzed for expression of Wt1 and SMA. A–C: After culture in normal medium, many peripheral cells of explants are fibroblastic and express SMA but not Wt1 (arrowheads), while Wt1-positive cells are negative for SMA (yellow open arrowhead). D–F: Under serum-free (sf) conditions, few of the epithelial sheet cells of cultured explants express SMA, whereas its majority express Wt1. SMA-positive cells can be Wt1-positive (open arrowheads) and Wt1-negative (arrowheads). G–L: Similar results were obtained for explants grown in sf-medium supplemented with basic fibroblast growth factor (bFGF) or epithelial growth factor (EGF), with few SMA-positive cells in the epithelial sheets. M–O: In explants grown in sf-medium supplemented with platelet-derived growth factor-BB (PDGF-BB), many fibroblastic peripheral cells in the epithelial sheet express SMA (arrowheads), many of which coexpress Wt1 (open arrowheads). Scale bar = 200 μm.

Article Snippet: Growth Factors Growth factors used were recombinant human EGF (40 ng/ml; R&D systems, 236-EG), recombinant human bFGF (40 ng/ml; R&D systems, 234-FSE/CF), and recombinant human homodimer PDGF-BB (40 ng/ml; Chemicon, GF018).

Techniques: Expressing, Cell Culture, Derivative Assay

Journal:

Article Title: Serosal Mesothelium Retains Vasculogenic Potential

doi: 10.1002/dvdy.21334

Figure Lengend Snippet: Production of smooth muscle actin (SMA) -positive cells in cultures of serosal mesothelium in the presence of specific growth factors. Cultures were analyzed at 5 days of culture with anti-SMA and anti-Wt1 antibodies. The total number of SMA-positive cells at the periphery was determined for each group indicated. One-way analysis of variance analysis determined that the number of SMA-positive cells in control serum-containing cultures was not significantly different from cultures with serum-free (sf) medium supplemented with platelet-derived growth factor-BB (PDGF-BB). In contrast to differentiation in serum-containing conditions, the number of SMA-positive cells in sf-medium or sf-medium supplemented with epithelial growth factor (EGF) or basic fibroblast growth factor (bFGF) was significantly (P < 0.001) reduced.

Article Snippet: Growth Factors Growth factors used were recombinant human EGF (40 ng/ml; R&D systems, 236-EG), recombinant human bFGF (40 ng/ml; R&D systems, 234-FSE/CF), and recombinant human homodimer PDGF-BB (40 ng/ml; Chemicon, GF018).

Techniques: Control, Derivative Assay

Journal: Cell stem cell

Article Title: A Non-Coding Disease Modifier of Pancreatic Agenesis Identified by Genetic Correction in a Patient-Derived iPSC Line

doi: 10.1016/j.stem.2020.05.001

Figure Lengend Snippet: Key Resources Table:

Article Snippet: Recombinant human bFGF , R & D Systems , 233-FB/CF.

Techniques: Recombinant, Knock-Out, Reporter Assay, Plasmid Preparation, Cloning, Quantitative RT-PCR, Software