Fig. 3.

Abi1 knockdown inhibited Bcr-Abl-stimulated actin cytoskeleton remodeling, MT1-MMP clustering, as well as cell adhesion and migration on fibronectin-coated surfaces. (A) Inhibition of Bcr-Abl-induced abnormal actin remodeling by Abi1 knockdown. Ba/F3 cells and the Ba/F3 cells expressing p185^wt, p185^K671R and p185^wt plus Abi1 shRNA were fixed and stained with TRITC-conjugated phalloidin. The cells with F-actin-rich structures (invadopodia-like structures) were visualized by fluorescence microscopy as shown by arrowheads (upper panel) and were counted (lower panel, represented as mean ± SD percentage of three randomly picked areas). (B) The p185^wt cells expressing GFP–MT1-MMP were transduced with either control retrovirus (control) or the retrovirus expressing Abi1 shRNA. The knock down of Abi1 expression was confirmed by western blotting (data not shown). The distribution of GFP–MT1-MMP was visualized by fluorescence microscopy. A similar result has been described previously (41). (C) Effects of Abi1 knockdown on Bcr-Abl-stimulated cell adhesion (lower panel) and migration (upper panel) on fibronectin-coated surfaces. Ba/F3 cells and the p185^wt cells transduced with either non-silencing shRNA or Abi1 shRNAs were grown in fibronectin-coated six-well plate (2.5 × 10⁵ per well) for 16 h. 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 ± SD of triplicate wells. For cell migration on fibronectin-coated membrane, 1 × 10⁵ cells were tested in Transwell migration assay. The vertical axis shows the percentage of the migrated cells and is expressed as the mean ± SD of triplicate wells; *P < 0.01.