All cells were grown either in DMEM or in RPMI-1640 and supplemented with 10% Selleckchem OSI-744 FCS plus antibiotics. The influence of BSc2118 on the growth of 22 tumor cell lines was analyzed using a crystal violet assay similarly as described for bortezomib by Adams et al [30]. GI50 is defined as the concentration needed to reduce the growth of treated cells to half that of untreated cells. Briefly, cells were seeded in quadruplicates on 96-well plates, exposed for 48 hours to proteasome inhibitors in 7 dilutions

ranging from 10 nM to 1000 nM (for BSc2118) and from 1 nM to 100 nM (for bortezomib). The cytostatic/cytotoxic effects of both BSc2118 and bortezomib on treated cells were compared to that of control cells. The mean viability for the whole cell panel was calculated in two ways, thereafter. First, average viability for the entire panel was calculated for each concentration point followed by calculation of the average GI50 value. Second, GI50 was averaged for each cell line individually. Both methods of calculation resulted in similar results. 20S proteasomes were isolated from both red blood cells of healthy volunteers and from murine organs [31]. Lysates from murine organs after injection of inhibitors were obtained by homogenization in 100 mM HEPES, pH 7.4, 2 mM MgCl2, 0.1% NP-40, 5 mM dithiothreitol and completed by Ultra-Turrax T8 ATM inhibitor (IKA-Werke). Chymotrypsin-like

activity of the 20S proteasome was measured with a fluorogenic substrate (Suc-LLVY-AMC, Bachem, Germany). Briefly, 100 ng of purified proteasomes

were exposed to proteasome inhibitors (0-1000 nM) and incubated with 50 μM of fluorogenic substrate for up to 60 min. Lysates normalized to protein content were directly incubated with 50 μM of fluorogenic substrate. The fluorescence was measured with POLARstar reader (BMG Labtech, Germany). The excitation and emission wavelengths were 390 nm and 460 nm, respectively. All experiments were performed in quadruplicates and repeated at least three times. Differences between groups were mafosfamide calculated by a Student’s t test. A P value of < 0.05 was considered to be statistically significant. For analysis of inhibitor stability in the presence of microsomal enzymes, BSc2118 and MG132 (at 0.1 to 5 μM, respectively) were incubated with mouse (Balb/c) microsomes (GIBCO) for up to 24 hours according to manufacturer instructions. The proteasome activity (20S isolated from mouse muscles) was measured in the presence of inhibitors with/without microsomal fraction as described in the section above. The results are displayed as relative 20S activity in the presence of inhibitors incubated with microsomal fraction. Inhibitors incubated with PBS were defined as 100%. The data are displayed as decrease of inhibitory activity in the presence of microsomal enzymes. Differences between groups were calculated by Student’s t test. A P value of < 0.05 was considered to be statistically significant.