HHT induced down-regulation of Mcl-1. (A) HHT induced down-regulation of Mcl-1 protein levels. CLL cells were incubated with 50, 100, 200, or 400nM HHT for 12 or 24 hours, when the cells were collected and lysed. Proteins such as PARP, Mcl-1, XIAP, and Bcl-2 were analyzed by immunoblotting. β-Actin was used as a loading control; * indicates nonspecific binding. (B) The down-regulation of Mcl-1 by HHT was significant in multiple samples. The Mcl-1 protein levels (mean ± SEM) were normalized to β-actin and quantitated in 5 (0.1μM HHT) and 3 (1μM HHT) patient samples. (C) Time course of HHT-induced reduction of Mcl-1 protein and apoptosis. CLL cells were incubated with HHT for 24 hours. Mcl-1 level (■) was quantitated by immunoblotting (n = 2) and cell death (●) was measured by annexin/PI every 2 hours (n = 6). (D) Sensitivity of CLL cells to HHT correlated with the basal Mcl-1 expression in each patient sample. Seven CLL samples were incubated with increasing concentrations of HHT for 24 hours. Cell death was measured by annexin/PI after 24 hours and correlated to basal Mcl-1 levels, quantitated by immunoblotting.