HHT did not inhibit transcription and was synergistic with SNS-032. (A) HHT did not inhibit transcription as did SNS-032. CLL cells were incubated with 0.1, 0.2, or 1μM HHT (●) or with 0.3 or 1μM SNS-032 (■) for 24 hours. The mRNA levels of Mcl-1 were measured by real-time RT-PCR, each performed in duplicate, and compared with time-matched controls. The 18s RNA was used as normalize for loading. Each data point represents the mean ± SEM of 3 patient samples. (B) HHT was synergistic with SNS-032 in killing CLL cells. CLL cells were incubated with 25, 50, 75, 100, 125, or 150nM HHT for 24 hours with 50, 100, 150, 200, 250, or 300nM SNS-032 or with a combination of HHT and SNS-032 at a ratio of 1:2. Apoptosis was detected by annexin V–PI staining. Each data point represents the mean of 4 patient samples. The combination index was calculated by CalcuSyn with the use of the median effect method. (C) Combination of HHT and SNS-032 deplete more Mcl-1 than single drug alone. CLL cells were incubated with 100nM SNS-032 or 50nM HHT alone or in combination. PARP and Mcl-1 proteins were analyzed by immunoblotting. Cell death was measured by annexin/PI staining. Expected cell death was calculated as described in “Methods.” Two representative results from 4 CLL samples were shown. Ctrl indicates control; SNS, SNS-032; S+H, combination of SNS-032 and HHT.