Journal: Molecular cancer research : MCR

Article Title: Oncofetal Chondroitin Sulfate Glycosaminoglycans are Key Players in Integrin Signaling and Tumor Cell Motility

doi: 10.1158/1541-7786.MCR-16-0103

Figure Lengend Snippet: A, U2OS cells were preincubated with rVAR2 and tested for their capacity to adhere to Fibronectin (FN), Collagen I (C-I), Collagen IV (C-IV), and Plastic (PL). Adherent cells were stained with Methylene Blue and quantified by reading absorbance at 650nm. B, MG63 cells were preincubated with rVAR2 and tested for their capacity to adhere to Fibronectin (FN), Collagen I (C-I), Collagen IV (C-IV), and Plastic (PL). Adherent cells were stained with Methylene Blue and quantified by reading absorbance at 650nm. C, RH30 cells were preincubated with rVAR2 and tested for their capacity to adhere to Fibronectin (FN), Collagen I (C-I), Collagen IV (C-IV), and Plastic (PL). Adherent cells were stained with Methylene Blue and quantified by reading absorbance at 650nm. D, MDA-MB-231 cells were preincubated with rVAR2 and tested for their capacity to adhere to Fibronectin (FN), Collagen I (C-I), Collagen IV (C-IV), and Plastic (PL). Adherent cells were stained with Methylene Blue and quantified by reading absorbance at 650nm. E, U2OS cells were preincubated with rVAR2 or rContrl and allowed to adhere to plastic. The phosphorylation of Src and Erk1/2 kinases in response to the adhesion event was monitored by Western.

Article Snippet: The cells were stimulated with 1-3% FBS, 5ug/ml FN (Sigma) or 10-80ug/ml fibronectin CS1 peptide (GeneArt) for the given timepoints.

Techniques: Staining, Phospho-proteomics, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Oncofetal Chondroitin Sulfate Glycosaminoglycans are Key Players in Integrin Signaling and Tumor Cell Motility

doi: 10.1158/1541-7786.MCR-16-0103

Figure Lengend Snippet: A, Titration of rVAR2 staining of U2OS cells in flow cytometry analysis. B, Analysis of downstream integrin signaling in rVAR2 or rContr treated U2OS cells in response to FBS stimulation. Figure shows effect on Src and p130Cas phosphorylation. C, Analysis of downstream integrin signaling in rVAR2 or rContr treated MG63 cells in response to FBS stimulation. Figure shows effect on Src. D, Analysis of downstream integrin signaling in rVAR2 or rContr treated MDA-MB-231 cells in response to FBS stimulation. Figure shows effect on Src. E, Analysis of downstream integrin signaling in U2OS cells treated with siRNA (72hrs) against B3GAT1 and CSGALNACT1 (CSGT-1). A scrambled siRNA is used as control. Figure shows effect on Src and p130Cas phosphorylation. F, RT-PCR analysis of cells treated with siRNA against B3GAT1 and CSGALNACT1 for the analysis shown in E. G, Analysis of FAK phosphorylation in rVAR2 or rContr treated U2OS cells in response to stimulation with the CS1 fibronectin peptide.

Article Snippet: The cells were stimulated with 1-3% FBS, 5ug/ml FN (Sigma) or 10-80ug/ml fibronectin CS1 peptide (GeneArt) for the given timepoints.

Techniques: Titration, Staining, Flow Cytometry, Phospho-proteomics, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cells

Article Title: Chemically Defined Xeno- and Serum-Free Cell Culture Medium to Grow Human Adipose Stem Cells

doi: 10.3390/cells10020466

Figure Lengend Snippet: Identifying attachment substrates necessary for human adipose stem cells’ (hASCs) adherence to cell culture surface, spreading, and growth under serum-free conditions. Human ASCs obtained from three different donors were used to screen a 96-well plate coated with 24 different peptides non-covalently linked to four different highly sulphated glycosaminoglycans (GAGs: synthetic dextran sulfate, heparin, chondroitin, and dermatan) [ , ]. The cells’ growth in the 96-well plate was evaluated using a commercial colorimetric cell viability assay. ( A ) Values of relative absorbance measured at 450 nm and classified according to the four types of sulphated GAGs (synthetic dextran sulfate, heparin, chondroitin, and dermatan) were used as a backbone for the formation of the different coating matrices. The four GAGs do not have a great influence on cell attachment and growth. ( B ) Ten different peptides non-covalently linked to highly sulphated GAGs strongly influence the ability of hASCs to attach and grow in the test micro-wells. The results obtained with the 14 poor-performing peptide-combinations are not shown. As in A, cell proliferation was measured using a commercial colorimetric kit. Histograms show the means ± standard deviations of the measured relative absorbances for hASCs obtained from three different donors. FN = fibronectin; VN = vitronectin; BS = bone sialoprotein; OP = osteopontin peptide; BMP2 = bone morphogenetic protein; PP = perlacan peptide; ColI = collagen I peptide; FGF = fibroblast growth factor.

Article Snippet: For this reason, we routinely culture hASCs on Corning PureCoatTM ECM Mimetic Fibronectin Peptide plasticware [ ], and all experiments of this study were performed using cells grown and expanded in serum-free conditions on this type of vessels.

Techniques: Cell Culture, Viability Assay, Cell Attachment Assay

Journal: eLife

Article Title: CCN1 interlinks integrin and hippo pathway to autoregulate tip cell activity

doi: 10.7554/eLife.46012

Figure Lengend Snippet: ( A ) Starved HUVECs were allowed to adhere to vitronectin (left) and fibronectin (right) after treated with CCN1 (10 ng/ml) or VEGF (10 ng/ml) in the presence or absence of integrin β3 monoclonal antibody (Ab) for 45 min, washed with PBS and stained with Crystal violet for 10 min, lysed with 0.1% SDS and measured absorbance (O.D.) at 550 nm. *p<0.01 vs. control group, **p<0.01 vs. no Ab group. ( B ) HUVECs were transfected with small interfering RNAs targeting ITGB3 and then treated with CCN1 (10 ng/ml) or VEGF (10 ng/ml) for 24 hr. Cell lysates were immunoblotted with an anti-integrin β3 antibody. ( C ) Cells were stained with antibodies against integrin β3 followed by a rhodamine-labelled secondary antibody after treated with CCN1 (10 ng/ml) or VEGF (10 ng/ml) for 24 hr. Nuclei were stained with DAPI. ( D ) Proximity ligation assay was performed and double fluorescent microscopic images showing protein-protein interactions in cells.

Article Snippet: Peptide recombinant protein , Vitronectin , Corning , 354238 , .

Techniques: Staining, Control, Transfection, Proximity Ligation Assay, Protein-Protein interactions

Journal: eLife

Article Title: CCN1 interlinks integrin and hippo pathway to autoregulate tip cell activity

doi: 10.7554/eLife.46012

Figure Lengend Snippet:

Article Snippet: Peptide recombinant protein , Vitronectin , Corning , 354238 , .

Techniques: Neutralization, Blocking Assay, Recombinant, Luciferase, Plasmid Preparation, Sequencing, Negative Control, Transgenic Assay, Mutagenesis, Software

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Fibronectin , STEMCELL Technologies , 07159 , .

Techniques: CRISPR, Knock-Out, Expressing, Construct, Transfection, Plasmid Preparation, Cell Culture, Stem Cell Culture, Recombinant, Selection, Blocking Assay, Control, Cloning, Incubation, Microscopy, Chemotaxis Assay, Software, Cell Tracking Assay