Figure 6. LCI as a real-time reporter of the dynamics of γ-secretase activity and inhibition in live cells.

A. NΔE/RBPjκ LCI reporter cells treated with different concentrations of the γ-secretase inhibitor DAPT exhibited a time- and dose-dependent decrease in complementation activity. B. Complementation activity correlated well with the amount of cleavage product NICD determined by Westerns. C. Kinetics of recovery after removal of inhibitors: All GSIs tested were confirmed to act in a reversible manner with slight differences in recovery half times. D. The NΔE/RBPjκ LCI reporter cells are robust and have been validated for high throughput screening applications. Multiple 96-well plates were seeded and treated with DMSO or DAPT and assayed for bioluminescence and viability. Z’ factors obtained in these experiments were >0.5, indicating that the assay is excellent for HTS. E. Knockdown by siGL3 Luc and siNCSTN was confirmed by Western analyses. siGL3 Luc efficiently targets Notch-NLuc. siNCSTN efficiently reduced NCSTN protein as well as mature γ-secretase complexes, indicated by the reduction in Presenilin1 NTF fragments. F. Luciferase complementation activity was greatly diminished with siGL3 Luc but not with siNCSTN, suggesting that γ-secretase is not limiting in these cells. G. siNCSTN sensitizes cells to sub-inhibitory concentrations of DAPT.