After differentiation for 6–8 weeks, 2D neurons were treated with either BACE1 [35], γ-secretase inhibitor Compound E [36] (Fig 6C) or vehicle (DMSO), and conditioning media were collected for quantification of Aβ by ELISA [37]. γ-Secretase inhibitor Compound E is a widely used potent inhibitor for many in vitro and in vivo studies. The half maximal inhibitory concentration (IC50) of Compound E in most in vitro γ-secretase activity assays is in the low nM range [36]. When our 2D neurons were treated with 0.1 μM Compound E (g-SI, Fig 7), all neurons produced significantly less Aβ40 and Aβ42. Interestingly, the reduction of Aβ40 and Aβ42 did not increase when higher doses (up to 1 μM) of Compound E were used (Fig 7). The efficacy of BACE1 inhibitor was obvious in 2D neurons (Fig 8). When neurons were exposed to 0.1–1 μM BACE1 inhibitor (BI, Fig 8), a significant reduction of Aβ40 and 42 was observed in all five lines (Fig 8). All Aβ40 levels decreased dramatically when higher concentrations of BACE1 inhibitor were applied. A similar pattern was observed for Aβ42.
