HHT inhibited protein synthesis. (A) HHT was a more potent inhibitor of protein synthesis than CHX. CLL cells were incubated with 0.1, 1, 10, or 100μM HHT (□) or CHX (○) for 24 hours. Then, 1 μCi/mL (3.7 × 10⁷ Bq/L) [³H]leucine was added to the culture for 1 hour, and radioactivity was determined by liquid scintillation counting (A). Apoptosis was detected by annexin V–PI staining (B). Each data point represents the mean ± SEM of 3 patient samples. Concentration-dependent PARP cleavage and loss of Mcl-1 were detected by immunoblotting (C). (D) Polysomal profiling analysis of CLL cells treated with HHT. CLL cells were incubated in HHT for 2 hours before lysed and resolved on a 10%-50% sucrose gradient. Twenty-two fractions were collected from each sample. The absorbance at 260 nm (OD260) was presented in the top panel. Solid line indicates control; dashed line, HHT treated. Mcl-1 and β-actin mRNA were quantitated in every other fraction and presented as percentage of total in all fractions. The analysis was repeated in 3 CLL samples with similar results. Results from one representative CLL sample were shown. Open bars indicate controls; solid bars, treatment with 200nM HHT.