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: 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

Journal: bioRxiv

Article Title: Multiple FGFR1 mutations modulate tumorigenic mechanisms in glioneuronal tumors

doi: 10.1101/2025.05.27.654799

Figure Lengend Snippet: R661P reduces K656E and N546K mutant protein stability by rescuing lysosomal degradation. A) Receptor stability experimental design: HEK293 cells expressing WT and mutant FGFR1 are stimulated with human FGF2 (hFGF2, 25 ng/mL) to induce receptor internalization and degradation and, in parallel, with cycloheximide (100 µg/mL) to block protein synthesis. B and D) Western blot analysis of a representative time-course experiment comparing WT FGFR1, K656E (B) and N546K (D) single and double mutants showing total Flag-FGFR1 protein levels at 0, 3 and 6 hours post-treatment with FGF2 and cycloheximide (CHX). C and E) Relative amounts of WT, single and double mutant FGFR1 protein obtained by quantifying western blots from n = 3 independent experiments. Values have been normalized (FC) against each relative 0h reference values. Data is represented by mean ± SEM and significant variations in protein amounts against each specific reference values have been indicated (ANOVA test, p value * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001) in the bar plots.

Article Snippet: Next, cells were induced with 25 ng/mL of recombinant human FGF2 (RD Systems) and treated with 100 µg/mL of Cycloheximide (Merck) and 200 nM of Bafilomycin A1 (Merck).

Techniques: Mutagenesis, Expressing, Blocking Assay, Western Blot

Journal: Stem cell research & therapy

Article Title: Differentiation of adipose-derived stem cells into Schwann cell-like cells through intermittent induction: potential advantage of cellular transient memory function.

doi: 10.1186/s13287-018-0884-3

Figure Lengend Snippet: Fig. 1 Identification of adipose-derived stem cells (ASCs) and schematic of the experimental design. a Primary ASCs grew in clusters and had a rounded spindle-like shape. b ASCs at passage 2 could differentiate into adipocytes, and the visual field in photographs was filled with red lipid droplets stained with Oil Red O solution. c ASCs differentiated into osteocytes formed calcium nodules with burrs, which were stained red by Alizarin Red solution. d In cartilage pellets, many cartilage lacunae were found, and the glycosaminoglycans around the chondrocytes were stained purple-blue by Toluidine Blue O solution. e Flow cytometry showed that more than 98% of ASCs were immunopositive for the MSC markers CD29, CD44, and CD90, while less than 6% of ASCs were immunopositive for the hematopoietic stem cell markers CD34, CD45, and CD86. f Schematic showing the experimental groups and induction methods. bFGF, basic fibroblast growth factor; β-ME, β-mercaptoethanol; dASC, differentiated ASC; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; SC, Schwann cell; uASC, undifferentiated ASC

Article Snippet: Pre-induction medium I was replaced with pre-induction medium II consisting of DMEM, 10% FBS, and 35 ng/ml all trans-retinoic acid (RA) (R2625; Sigma) and incubated for 72 h. The resulting differentiated ASCs were divided into four groups according to subsequent processing: (ii) sustaining dASCs 4d: Differentiated ASCs (dASCs) were induced for 4 days with complete induction medium consisting of DMEM, 10% FBS, 5 μM forskolin (F6886; Sigma), 200 ng/ml recombinant human heregulin-β1 (HRG) (100–03; PeproTech), 10 ng/ml basic fibroblast growth factor (bFGF) (3339-FB: R&D Systems), and 5 ng/ml recombinant rat plateletderived growth factor (PDGF)-AB (1115-AB; R&D Systems); (iii) sustaining dASCs 7d: dASCs were induced for 7 days with complete induction medium; (iv) sustaining dASCs 10d: dASCs were induced for 10 days with complete induction medium; (v) intermittent dASCs: dASCs were induced for 4 days with complete induction medium, after which complete induction medium was replaced with incomplete induction medium consisting of DMEM, 10% FBS, 5 μM forskolin, and 200 ng/ml HRG for induction for 3 days.

Techniques: Derivative Assay, Staining, Flow Cytometry, Modification

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