Cell adhesion–mediated down-regulation of Bim is required for cell adhesion–mediated drug resistance. (A-B) Overexpression of Bim inhibits cell adhesion–induced Bim down-regulation, induces cell apoptosis, and overcomes CAM-DR. Jeko-1 lymphoma cells were transfected with pCMV-SPORT6 control (pl.Ctrl)- or pCMV-SPORT6-Bim (pl.Bim) for 24 hours. Cells, collected and washed in suspension versus in adhesion with HK, were treated with mitoxantrone (1μM) for another 12 hours; Bim protein levels (A) were then analyzed by Western blot, and apoptosis (B) was analyzed by annexin V with flow cytometry. Data are representative of 4 independent experiments. In panel A, the relative change in Bim protein was measured by quantitative densotometry and is indicated below each lane. (C) Bim knockdown by shRNAs in Jeko-1 and SUDHL-4 cells. Bim expression levels in Jeko-1 cells were analyzed by Western blot after 48 hours of transfection with either Bim shRNA constructs 1 or 2 or 3 or 4 (shBim construct 1 or 2 or 3 or 4) or nonsilencing control shRNA (shCtrl-nonsilencing). Results are representative of 3 independent experiments. (D-E) Knockdown of Bim inhibits mitoxantrone-induced cell apoptosis in Jeko-1 and SUDHL-4 cells in the presence and absence of HK adhesion. Jeko-1 (D) or SUDHL-4 (E) cells transfected with Bim shRNA (shBim-3) or control shRNA (shCtrl) were incubated for 48 hours and then treated with vehicle control (VC) or mitoxantrone (0.2μM) for another 24 hours. Apoptosis was analyzed by flow cytometry using annexin V. Data are representative of 4 independent experiments.