Overexpression of CrkL increases adhesion of 32D cells. (A) Morphology of 32D cells overexpressing CrkL. A clone of 32D/EpoR-Wt cells stably transfected with the expression plasmid for tetracycline transactivator alone (32DE/TA) or a clone also transfected with pTet-CrkL (32DE/Tet-CrkL) was cultured in the presence (+) or absence (−) of 100 ng/mL of tetracycline (Tet), as indicated, for 24 hours and photographed under an inverted microscope (Olympus, Tokyo, Japan). (B) Adhesion of CrkL-overexpressing 32D cells to fibronectin. 32DE/TA and 32DE/Tet-CrkL cells were cultured in the presence (+) or absence (−) of Tet, as indicated, for 24 hours and allowed to attach to wells coated with 10 μg/mL fibronectin for 30 minutes at 37°C in the presence of IL-3. The extent of cell adhesion was quantitated as described in Materials and Methods. The data represent averages ± SD of triplicate determinations. Anti-CrkL immunoblotting of cell lysates obtained under the same conditions is also shown. (C) Effect of fibronectin concentration on adhesion of CrkL-overexpressing 32D cells. 32DE/Tet-CrkL cells, cultured with or without tetracycline, as indicated, were allowed to attach to wells coated with indicated concentrations of fibronectin for the cell adhesion assay. (D) Effect of cytokines on adhesion of CrkL-overexpressing 32D cells. 32DE/Tet-CrkL cells were cultured with or without tetracycline, as indicated, for 24 hours. During the last 16 hours, cells were cultured with 1 U/mL Epo (Epo), 5 ng/mL IL-3 (IL-3), or without any cytokine (−), as indicated. Cells were allowed to attach to wells coated with 10 μg/mL fibronectin for the cell adhesion assay in the presence or absence of cytokine, as indicated.