Cotreatment with bortezomib/flavopiridol synergistically induces apoptosis in multiple Bcr/Abl^-human leukemia cells, accompanied by abrogation of NF-κB/DNA binding activity and activation of JNK. (A) U937 cells were exposed to 5 nM bortezomib (Btzmb or Bz) ± 100 nM flavopiridol (FP) for 24 hours, after which the percentage of cells exhibiting apoptotic morphology and reduced Δψm was determined by evaluating Wright Giemsa-stained cytospin preparations and monitoring DiOC₆ uptake, respectively. Results represent the means ± SDs for 3 separate experiments performed in triplicate. (B) Alternatively, cytosolic (S-100) fractions and whole-cell lysates were prepared, and expression of cytochrome c and Smac/DIABLO in cytosol (upper panels) and PARP cleavage (lower panels) was monitored by Western blot. CF indicates cleavage fragment. Each lane was loaded with 30 μg protein; blots were stripped and reprobed with antiactin antibodies to ensure equal loading and transfer of protein. An additional 2 studies yielded equivalent results. (C) U937 cells were treated as described for panel A, after which nuclear extracts and cell lysates were prepared and subjected to electrophoretic mobility shift assay (EMSA, top blot) and Western blot analysis (WB, lower blots), respectively. Results are representative of 3 separate experiments. (D) Multiple human leukemia cells were exposed to bortezomib (Btzmb or Bz, HL-60, 3 nM; Jurkat, 7.5 nM; Raji and CCRF-CEM, 5 nM) ± flavopiridol (FP, 100 nM) for 24 hours, after which the percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright Giemsa-stained cytospin preparations. Results represent the means ± SDs for at least 3 separate experiments performed in triplicate.