Effect of 17-AAG on cytarabine-induced cell-cycle arrest and cytotoxicity in ML-1 cells. (A-B) ML-1 cells were treated for 24 hours with diluent or 30 nM cytarabine (Ara-C). Either 300 nM 17-AAG (A) or the indicated concentration of 17-AAG (B) was then added for an additional 24 hours in the continued presence of cytarabine. At the completion of the drug treatment, cells were analyzed by flow microfluorimetry as illustrated in Figure 2. Arrows in panel A indicate subdiploid (apoptotic) cells. (C) ML-1 cells were treated with diluent or 30 nM cytarabine for 24 hours, washed, and incubated with diluent, 300 nM 17-AAG, 30 nM cytarabine, or 30 nM cytarabine plus 300 nM 17-AAG for an additional 24 hours. Morphologic apoptotic changes were then assessed as illustrated in Figure 4C. (D-F) ML-1 cells were treated with diluent or 100 nM cytarabine for 24 hours, sedimented, and incubated with diluent, 300 nM 17-AAG, 100 nM cytarabine, or 100 nM cytarabine plus 300 nM 17-AAG for an additional 24 hours. At the completion of the incubation, whole-cell lysates were assayed for ability to cleave DEVD–rhodamine 110 and for DNA content (D). Alternatively, whole-cell lysates were subjected to immunoblotting using sera that recognize the indicated antigen (E); dashed line indicates where nonadjacent lanes on each blot were juxtaposed; arrow, 89 kDa PARP fragment that results from caspase-mediated cleavage.⁸⁶  At the completion of the incubation, cells were also examined for APC–annexin V binding (F) as illustrated in Figure 4E. Error bars: ± 1 standard deviation of 5 (C) or 3 (F) independent experiments. ^*P = .02 and ^**P < .001 by paired t test.