human plasma fibronectin (fn)  (Millipore)

Journal: Communications Biology

Article Title: A proteomics approach to isolating neuropilin-dependent α5 integrin trafficking pathways: neuropilin 1 and 2 co-traffic α5 integrin through endosomal p120RasGAP to promote polarised fibronectin fibrillogenesis in endothelial cells

doi: 10.1038/s42003-024-06320-4

Figure Lengend Snippet: Internalised NRP-α5 integrin complexes undergo Rab11 + long-loop recycling via p120RasGAP, promoting polarised nascent adhesion assembly and directional migration in endothelial cells. In the absence of NRP1 and NRP2, α5 integrin arrests in early endosomes and Rab7 + late endosomes, before being recycled via retrograde transport and the dynein–dynactin complex. In vivo, endothelial deletion of NRP1 and NRP2 severely impairs polarised sprouting and fibronectin deposition. Created with BioRender.com.

Article Snippet: Cell pellets were seeded onto plasticware coated with a solution of 0.1% gelatin containing 10 μg/ml human plasma fibronectin (FN) (Millipore). and collagen type 1. mLMECs were twice positively selected for using endomucin primary antibody and magnetic activated cell sorting (MACS) as previously described by Reynolds & Hodivala-Dilke , prior to immortalisation using polyoma-middle-T-antigen (PyMT) as previously described by Robinson et al. . ECs were cultured in a 1:1 mix of Ham’s F-12:Dulbecco’s Modified Eagle Medium (DMEM) (low glucose) medium supplemented with 10% foetal bovine serum (FBS), 100 units/mL penicillin/streptomycin (P/S) and 50 μg/mL heparin (Sigma) at 37 °C in a humidified incubator (+5% CO 2 ) unless otherwise stated.

Techniques: Migration, In Vivo

Journal: Breast Cancer Research : BCR

Article Title: A novel role for signal transducer and activator of transcription 5b (STAT5b) in β 1 -integrin-mediated human breast cancer cell migration

doi: 10.1186/bcr2341

Figure Lengend Snippet: STAT5b knockdown inhibits β1-integrin-mediated migration to fibronectin (FN). (a) The undersides of trans-well filters were coated with 10 μg/ml FN or vitronectin (VN) overnight at 4°C. BT-549 and MDA-MB-231 cells were placed in serum-free media in upper chambers, and 1% fetal bovine serum (FBS) (BT-549) or 10% FBS (MDA-MB-231) medium was placed in the lower chambers for FBS controls. Serum-free medium was placed in lower chambers for FN and VN conditions. Migration was allowed to proceed for 3 hours (BT-549) or 6 hours (MDA-MB-231) (n = 3). (b) BT-549 and MDA-MB-231 cells were pretreated for 1 hour at 37°C with 10 μg/ml β 1 -integrin-blocking antibody or DMSO control (con). Cells were plated in trans-well chambers in the presence of blocking antibody, and migration to 1% FBS (BT-549) or 10% FBS (MDA-MB-231) was measured. Student's t test was used to determine statistical significance ( P < 0.05) between the following: BT-549: con and antibody (*) (n = 6); MDA-MB-231: con and antibody (*) (n = 4). (c) The undersides of trans-well filters were coated with 3 μg/ml FN overnight at 4°C, and migration assays were performed with siRNA-transfected cells as described in part a. One-way ANOVA with Tukey's post-test was used to determine statistical significance ( P < 0.05) between the following: BT-549: siLuc and siSTAT5b FBS (*), siLuc and siSTAT5b FN (black circles); n = 4. MDA-MB-231: siLuc and siSTAT5b FBS (*), siLuc and siSTAT5b FN (black circles); n = 3.

Article Snippet: For migration to extracellular matrix components, the undersides of filters were coated with 0 to10 μg/ml human plasma fibronectin (FN) (BD Biosciences) or recombinant human vitronectin (VN) (R&D Systems) overnight at 4°C, as indicated, and DMEM/0.1% BSA was used in both the upper and lower chambers.

Techniques: Migration, Blocking Assay, Transfection