Journal:

Article Title: Integrin ? 4 ? 1 Regulates Migration across Basement Membranes by Lung Fibroblasts

doi: 10.1164/rccm.200301-041OC

Figure Lengend Snippet: Connecting segment-1 (CS-1) peptide ligation with α4β1 integrin attenuates basement membrane migration in NLFs but not FLFs. (A) Matrigel transwell assay of NLFs in response to SF media alone, SF media with the 110-kD fibronectin fragment (110 kD), or a combination of the 110 kD and CS-1 containing fibronectin fragments (110 kD/CS-1). Cell migration is reported as mean number of cells per hpf ± SEM. The CS-1 fibronectin fragment attenuates migration across basement membranes by NLFs but not by FLFs. Migration across basement membranes by FLFs was not different between the 110-kD and the 110-kD/CS-1 groups. Data are representative of experiments with fibroblasts from five patients in each group. Each patient’s fibroblasts were assessed at least twice. (B) Fibronectin-induced migration across basement membranes is inhibited by α4β1 ligation. NLFs migrated across basement membranes minimally in response to intact fibronectin when treated with nonspecific IgG (control). In the presence of anti-α4 integrin antibody, intact fibronectin induced significant fibroblast migration across basement membranes.

Article Snippet: Cells were resuspended at a concentration of 1 × 10 6 cells/ml in FA buffer and were incubated with R-phycoerythrin–conjugated mouse anti-human α 4 integrin (Cymbus Biotechnology, Chandlers Ford, Hants, UK), fluorescein isothiocyanate–conjugated mouse anti-human α5 integrin (Cymbus Biotechnology) or the appropriately labeled IgG control antibody (BD Pharmingen, San Diego, CA) on ice.

Techniques: Ligation, Migration, Transwell Assay

Journal:

Article Title: Integrin ? 4 ? 1 Regulates Migration across Basement Membranes by Lung Fibroblasts

doi: 10.1164/rccm.200301-041OC

Figure Lengend Snippet: Cell-surface expression of the α4 and α5 integrin subunits on NLFs and FLFs. Equal numbers of each fibroblast line were incubated with fluorescein isothiocyanate–conjugated anti-α5 integrin antibody, R-phycoerythrin (PE)-conjugated anti-α4 integrin antibody, or the appropriate fluorochrome-labeled nonspecific IgG and assessed for fluorescence using flow cytometry. In all cases, each patient’s lung fibroblasts expressed both the α4 and α5 integrin subunits on the cell surface. Thin dotted lines represent nonspecific IgG staining, and solid lines represent the integrin-specific fluorescent signal. Data are representative results from experiments with fibroblasts from five patients in each group. To ensure reproducibility, each patient’s fibroblasts were assessed at least twice. UIP = usual interstitial pneumonia.

Article Snippet: Cells were resuspended at a concentration of 1 × 10 6 cells/ml in FA buffer and were incubated with R-phycoerythrin–conjugated mouse anti-human α 4 integrin (Cymbus Biotechnology, Chandlers Ford, Hants, UK), fluorescein isothiocyanate–conjugated mouse anti-human α5 integrin (Cymbus Biotechnology) or the appropriately labeled IgG control antibody (BD Pharmingen, San Diego, CA) on ice.

Techniques: Expressing, Incubation, Labeling, Fluorescence, Flow Cytometry, Staining

Journal:

Article Title: Integrin ? 4 ? 1 Regulates Migration across Basement Membranes by Lung Fibroblasts

doi: 10.1164/rccm.200301-041OC

Figure Lengend Snippet: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) lipid phosphatase activity is induced by CS-1 fibronectin fragment ligation of the α4β1 integrin. Human lung fibroblasts (IMR-90) were incubated for 18 hours with SF media alone, SF media containing 20-nM CS-1 peptide alone, or in the presence of nonspecific IgG or anti-α4β1 integrin antibody. Cells were assayed for PTEN activity, and results were expressed as the mean change in phosphate released (± SEM) for each condition, as compared with values obtained at baseline. PTEN lipid phosphatase activity is significantly induced by CS-1 ligation. This effect is specific for ligation of CS-1 with the α4β1 integrin. Changes in PTEN activity were not observed in the absence of ligands (data not shown). Results are representative of a duplicate set of experiments. (B) PTEN activity is induced in NLFs but decreased in FLFs in response to CS-1 peptide. NLFs (n = 5; open bar) and FLFs (n = 5; closed bar) were exposed to 20-nM CS-1 peptide, and PTEN immunoprecipitates were assayed for phosphatase activity. NLFs demonstrated increased PTEN activity, whereas FLFs decreased PTEN activity under the same conditions. Results are representative of the mean change in phosphate released by NLFs and FLFs.

Article Snippet: Cells were resuspended at a concentration of 1 × 10 6 cells/ml in FA buffer and were incubated with R-phycoerythrin–conjugated mouse anti-human α 4 integrin (Cymbus Biotechnology, Chandlers Ford, Hants, UK), fluorescein isothiocyanate–conjugated mouse anti-human α5 integrin (Cymbus Biotechnology) or the appropriately labeled IgG control antibody (BD Pharmingen, San Diego, CA) on ice.

Techniques: Activity Assay, Ligation, Incubation