the ice-cold ReN cell differentiation medium (1:2 dilution ratio) and vortexed with ReN cell pellets for 20 seconds. The final cell concentration for the mixture was approximately 1 × 107 cells/ml. 400 μl of the cell/Matrigel mixtures were immediately transferred into tissue culture inserts (ThinCerts, 0.4 μm pore size, Greiner Bio-One, Monroe, NC, USA) and then placed in 24-well plates (BD Biosciences). After 1 hour incubation at 37°C, 1 ml of the pre-warmed differentiation media was added and the cultures were maintained for 4–12 weeks; media was changed every 3–4 days. For drug treatments, differentiation media containing either 1 μM BACE1 inhibitor IV, 1 μM DAPT, 3.7 nM Compound E or the same volumes of DMSO were added to 4–6 week differentiated 3D-cultured ReN cells and then maintained for additional 2–3 weeks. The cells were either fixed with 4% paraformaldehyde (PFA) or harvested for extraction and WB analysis
