H9 hesc

Neural progenitor cell (NPC) differentiation was performed as previously described [37 ]. Dishes for hESC cultures were coated with 0.15 mg/mL growth factor reduced Matrigel (Corning Inc, 354230, Corning, NY, USA) in DMEM/F-12 (Thermo Fisher Scientific, 11320033) for 1 h at RT prior to cell seeding. H9 hESCs (WiCell, WA09, Madison, WI, USA) were seeded as colonies and maintained in mTeSR Plus (StemCell Technologies, 05825). NPC differentiation was initiated when the hESC cultures reached 90% confluence, by replacing growth medium with Knockout Serum Replacement (KSR) medium (414 mL Knockout-DMEM (Thermo Fisher Scientific, 10829018), 75 mL Knockout-serum replacement (Thermo Fisher Scientific, 10828028), 5 mL Glutamax (Thermo Fisher Scientific, 35050061), 5 mL MEM-NEAA (Thermo Fisher Scientific, 11140050), 500 µL 2-mercaptoethanol (Thermo Fisher Scientific, 21985023), 500 µL Gentamicin (Wisent Bioproducts 450-135), 10 µM SB431542 (Tocris, 1614, Bristol, UK) and 500 nM LDN-193189 (Stemgent 04-0074, Reprocell Inc, Yokohama, Kanagawa 222-0033, Japan). Differentiation medium was replaced daily on days 4 and 5 by 75:25 KSR:N2 medium (486.5 mL DMEM/F-12 (Thermo Fisher Scientific, 11320033), 5 mL 15% glucose, 5 mL N2 supplement (Thermo Fisher Scientific, 17502048), 500 µL 20 mg/mL human insulin (Wisent Bioproducts, 511-016-CM), 2.5 mL 1 M HEPES (Thermo Fisher Scientific, 15630080), 500 µL Gentamicin), on days 6 and 7 by 50:50 KSR:N2, on days 8 and 9 by 25:75 KSR:N2 and on days 10 and 11 by N2 medium containing 500 nM LDN-193189. On day 12, differentiated NPCs were treated with Y-27632 (Tocris, 1254) for 4 h, dissociated with Accutase (Stemcell Technologies, 07922) and seeded into Matrigel coated dishes containing Neural Induction Medium (NIM, 244 mL DMEM/F12, 244 mL Neurobasal medium (Thermo Fisher Scientific, 21103049), 2.5 mL N2 supplement, 5 mL B-27 Supplement (Thermo Fisher Scientific, 17504044), 2.5 mL Glutamax (Thermo Fisher Scientific, 35050061), 125 µL 20 mg/mL human insulin, 500 µL 20 µg/mL FGF2 (StemBeads, SB500, Rensselaer, NY, USA), 10 µL 1 mg/mL hEGF (Millipore Sigma E9644) and 500 µL Gentamicin) for expansion. NPCs were passaged at full confluence a minimum of one time before neuronal differentiation.
For NPC-neuronal differentiation culture dishes were coated with 0.001% Poly-L-ornithine (Millipore Sigma, P4957) at 4 °C overnight, followed by 25 µg/mL laminin (Millipore Sigma, L2020) for 2 h at room temperature. NPCs were treated with Y-27632 for 4 h, dissociated with Accutase and seeded at a density of 20,000 cells/cm² in NIM. Neuronal differentiation was initiated when NPCs reached 70% confluence by replacing growth medium with neuronal differentiation medium (244 mL DMEM/F-12 medium, 244 mL Neurobasal medium, 2.5 mL N2 supplement, 5 mL B27 supplement, 200 µL 50 µg/ml BDNF (Peprotech, 450-02), 200 µL 50 µg/ml GDNF (Peprotech 450-10, Thermo Fisher Scientific), 250 mg dibutyryl cyclic-AMP (Millipore Sigma, D0627), 500 µL 100 M L-ascorbic acid (FujiFim Wako Chemicals, 323-44822, Osaka, Japan), and 500 µL Gentamicin). Cells were fed every 3 days for 18 days to obtain immature neuronal networks. FMF-04-159-2 was dissolved in DMSO (Fisher Scientific, BP231) and applied in cell culture medium to hESC-derived neurons for 6 days (with a replenishment of FMF-04-159-2 -containing medium after the first 3 days) and to HEK293T cells for 18 h. HEK293T cells were treated with 500 nM of FMF-04-159-2 together with 200 nM of Bafilomycin A1 (in DMSO, NEB, 54645S) or 1 µM of MG-132 (in DMSO, EMD Millipore, 474790) for 18 h to inhibit autophagy or the UPS, respectively. For protein analysis cells were washed with cold PBS, scraped, and collected in low protein binding microcentrifuge tubes (Thermo Scientific, 90410). Cells were pelleted by centrifugation at 1000 × g for 5 min at 4 °C. The supernatant was aspirated, and the cells were lysed in cold RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate; 1% NP-40, 5 mM EDTA, pH 8.0) with protease and phosphatase inhibitors. Cell lysates were incubated on ice for 20 min, with vortexing every 5 min. Lysates were centrifuged at 18,000 × g for 20 min at 4 °C to pellet cell debris. Autophagic activity was monitored by immunoblots for LC3B (Novus Biologicals, NB100-2220). Proteasome activity was monitored by immunoblots for poly-ubiquitinated proteins (anti-ubiquitin P4D1, Cell Signaling Technology, 3136).