Figure 4

Examination of real-time γ-secretase activity in A20 lymphoma and in primary B-CLL patient samples. (a) Correlation between real-time inhibition of γ-secretase activity and GSI-mediated inhibition of A20 mouse lymphoma proliferation. Two 96-well plates were seeded with 50,000 A20 mouse lymphoma cells per well in 100 μl RPMI media. To each of these plates an additional 100 μl of media was added containing DMSO or GSI-34 to indicated final concentration. These plates were incubated for 48 hours at 37°C. Following this incubation, one plate was used in a real-time exo-cell assay to quantitate the real-time inhibition of γ-secretase in A20 cells. Briefly, A20 cells were pelleted and media removed. Fresh media containing 1 μM Sb4 substrate and 0.25% CHAPSO detergent were added and the exo-cell assay was performed. For each assay point n = 4, and s.d. is plotted. Additionally, to the other 96-well plate 2 μCi/ml [³H]thymidine was incubated with the cells for 5 hours. Following 5-hour incubation at 37°C, the amount of tritiated DNA was quantified on a β-counter. For each proliferation assay point n = 10, and s.d. is plotted. (b) Real-time γ-secretase activity in primary B-CLL patient samples. B-CLL cells were seeded in 96-well plate at a concentration of 50,000 cells per well. These were allowed to attach overnight. Subsequently, the media was removed and fresh media was added back that contained either DMSO or 1 μM Compound E inhibitor. This was incubated for 24 hours at 37°C. Cells were then washed once in PBS and exo-cell assay was performed as previously described. For each assay point n = 4, and s.d. is plotted.