WP1130 induces rapid ubiquitination and apoptosis in imatinib-sensitive and -resistant CML cells. (A) BV-173 (left) and BV-173R (right) cells were treated with the indicated compound for 72 hours before cell viability and proliferation was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide assays. The results represent the average ± SD of 4 replicates. Similar results were obtained in 2 additional independent studies. (B) BV-173R cells were treated with the indicated compound for the interval noted before cell lysates were immunoblotted for caspase 3, caspase 9, and PARP. The major cleaved forms of these proteins are also shown. (C) K562 cells were treated as noted (cells treated with the combination of agents were treated with 17-AAG followed by WP1130) before detergent-soluble cell lysates were prepared for direct Bcr-Abl blotting (top). Lysates were also subjected to Bcr-Abl or Hsp90 immunoprecipitation, followed by immunoblotting as noted. (D) K562 cells were treated as noted before analyzing the Hsp70, Hsp90, and ubiquitin content in the detergent-soluble and -insoluble fractions (fractions were resolved as described in lysate preparation section) by immunoblotting. Equal volumes of detergent-soluble and -insoluble extract were loaded in each lane. Hsp90 blotting served as a protein-loading control for the detergent-soluble fraction.