Fig. 8. Blockade of Abi1 pathway or 1-integrin function impaired the ability of Bcr-Abl to stimulate cell adhesion to fibronectin. (A) The Abi1 pathway is required for Bcr-Abl-stimulated cell adhesion to fibronectin-coated surfaces. Left panel: Ba/F3 cells and the Ba/F3 cells expressing either p185^wt or p185^SH3C were grown in fibroncetin-coated six-well plates (2.5x10⁵/well) for 16 hours. The total cells and the cells that were adherent to fibronectin-coated surfaces were counted and the percentage of adherent cells calculated. The vertical axis shows the percentage of the adherent cells and is expressed as the mean ± s.d. of duplicate wells. Data are representative of two independent experiments. Right panel: the Ba/F3 cells expressing p185^wt alone or p185^wt plus Abi1^PPLL were grown in fibroncetin-coated plates (2.5x10⁵/well) for 16 hours. The total cells and the cells that were adherent to fibronectin-coated surfaces were counted and the percentage of adherent cells calculated. The data represent the mean ± s.d. of triplicate wells. (B) The F-actin-rich structures are enriched in adherent p185^wt-transformed Ba/F3 cells. The p185^wt-transformed Ba/F3 cells were grown in fibronectin-coated plates with or without fibronectin supplemented in medium (5 µg/ml) for 16 hours. Adherent and non-adherent cells were harvested separately and stained by TRITC-conjugated phalloidin and DAPI to visualize F-actin and nuclei, respectively, by fluorescence microscopy (upper panel). The percentage of the cells containing the F-actin-rich structures (spots) from three randomly selected fields in adherent cells as well as non-adherent cells was statistically calculated and expressed as the mean ± s.d. (lower panel). The data are representative of three independent experiments. (C) Bcr-Abl-stimulated cell adhesion to fibronectin is 1-integrin dependent. The p185^wt-transformed Ba/F3 cells were treated with either Ha2/5 or a control hamster IgM (4 µg/2.5x10⁵ cells) and plated in fibroncetin-coated plates (2.5x10⁵ cells/well) for 16 hours. The total cells and the cells that were adherent to fibronectin-coated surfaces were counted and the percentage of adherent cells calculated. The data represent the mean ± s.d. of triplicate wells and are representative of two independent experiments.