Af 100 15 100

hNPCs derived from an H1 ESC line (WA01, WiCell Research Institute) were used in the experiments. ESCs were transduced with a viral construct containing red fluorescent protein and the EGFP and dsRed genes were inserted at the AAVS1 locus to generate fluorescence-expressing lines (Xing et al., 2019 (link)). Generation of NPCs from these ESC lines by using the monolayer culture method has been described previously (Xing et al., 2019 (link)). Briefly, when ESCs reached approximately 95% confluence, the culture medium was changed to N2B27 medium with 5 μM SB431542 (Selleck, S1067) and 5 μM dorsomorphin (Selleck, S7840) added for 8 days. The cells were then scraped down and replated in the N2B27 medium for another 8 days. hNPCs were obtained by manually picking rosettelike structures and maintained in suspension in the N2B27 medium with 20 ng/mL basic fibroblast growth factor (bFGF) (Peprotech, 9610018B50) and 20 ng/mL epidermal growth factor (EGF) (Peprotech, AF10015100).

Panobinostat (S1030) and BEZ235 (S1009) were purchased from Selleck Chem (Houston, TX, USA). U87, U251 and 293T cell lines were obtained from Cell Bank of Chinese Academy of Science (Shanghai, China). U87, U251 and 293T were cultured in Dulbecco’s modified Eagle medium/high glucose (HyClone, Logan, Utah, USA) with 10% fetal bovine serum. U87_Serum_Free were cultured using NeuroCult Neural Stem and Progenitor Cells (NS-A) Proliferation Kit (Human) (05751, Stem Cell Technology, Vancouver, Canada) supplemented with human epidermal growth factor (EGF) basic (20 ng/ml) (AF-100-15-100, PeproTech, New Jersey, USA), human fibroblast growth factor (FGF) basic (20 ng/ml) (100-18B-100, PeproTech), and 0.2% heparin solution (10 ng/ml) (07980, Stem Cell Technology) and passaged for 1.5 months. Mouse neural stem cells (mNSCs) were established from fresh mouse brain tissues. Briefly, neonatal mouse brains were dissected, and the cerebral cortex was dissociated into single cells with Gibco TrypLE Express Enzyme (12604021, Thermo Fisher Scientific, Waltham, USA) for 10 min at 37°C. Excess debris were removed with 22% Percoll solution and mNSCs were cultured in NeuroCult NS-A Proliferation Kit (Mouse) (05702, Stem Cell Technology) supplemented with murine EGF (20 ng/ml) (315-09-100, PeproTech), murine-FGF (20 ng/ml) (450-33-50, PeproTech) and heparin (10 ng/ml).