Proliferation Assays and Colony Formation

Cell proliferation was measured by CCK-8/WST-1 or EdU assays. For EdU assays, we used the Click-iT™ Plus EdU Cell Proliferation Kit for Imaging (Invitrogen). After EdU incubation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100, and EdU detection was performed according to the manufacturer’s instructions. Nuclei were counterstained with Hoechst 33342 reagent. At least 500 nuclei were counted in triplicate, and the number of BrdU-positive nuclei was recorded. For colony-formation assays, Ctrl and shRNA-infected cells were seeded in six-multiwell plates. After 2 weeks, cells were fixed with 4% PFA and stained with crystal violet. For neurospheres, 1000 cells/ml were seeded in low attachment 96-multiwell plates in DMEM with neurosphere medium as previously described^{13 (link),89 (link)}. The number of neurospheres was counted and captured images after 10–15 days.