BFL-1 and/or MCL-1 proteins are up-regulated in resistant cell lines and are inducible upon treatment with ABT-737. (A) Immunoblot analysis of OCI-LY1, OCI-LY1 R7, and OCI-LY1 R10 cell line lysates with the indicated antibodies. − indicates that ABT-737 was withdrawn for 3 weeks; +, continuous culture with ABT-737. Unpictured antibody controls confirmed the efficacy of the BCL-XL and BFL-1 antibodies used in this assay. These results are representative of 3 independent experiments. (B) OCI-Ly1 R7 cells were removed from ABT-737–containing media for 3 weeks. ABT-737 was added back to culture for the indicated time frame at 320nM. Whole-cell lysates were made after treatment and analyzed by immunoblot. DMSO was used as a solvent-only negative control. These results are representative of 3 independent experiments. (C) SU-DHL-4 R2 cells were removed from ABT-737–containing media for 3 weeks. SU-DHL-4 and SU-DHL-4 R2 cells were treated with 1μM ABT-737 or DMSO for 12 hours after pretreatment with 10μM ZVAD.fmk or DMSO for 1 hour as indicated. Whole-cell lysates were made after treatment and analyzed by immunoblot. These results are representative of 3 independent experiments. ND indicates that these samples were not analyzed.