Fig. 6

Reduction in lysosomal activity and slow induction of quiescence by Tfeb-knockout. a Immunoblot of TFEB in Tfeb-KO NSCs (HOMO) and control NSCs (HET) in the quiescent state in vitro. The parental cells (without Cre excision) and tdTomato-positive cells (purified by cell sorting after Cre excision) were analyzed after culture in quiescence medium. b Immunocytochemistry of TFEB (green) in Tfeb-KO qNSCs and control qNSCs. Cre-excised cells were labeled with tdTomato (red). c Bright field and LysoTracker-green staining in Tfeb-KO and parental control NSCs in the quiescent state. d mRNA expression of lysosomal genes in Tfeb-KO NSCs (gray bars) and controls (white bars) in the quiescent state (n = 4). e Representative immunostaining 3 days after BMP exposure. f Proliferation of Tfeb-KO (gray bars) and control NSCs (white bars) in quiescence medium. Cells were fixed, stained with Sox2 (blue) and Ki-67 (green) at 1 and 3 days following BMP exposure, and analyzed (n = 12). g Representative immunoblot for EGFR, Notch1, and their active forms in Tfeb-KO and control in proliferation medium (aNSC) and on 1 and 3 days in quiescence medium. h Relative quantification of P-EGFR, EGFR, NICD, and Notch1 on 1 day after BMP exposure in g (n = 4). i Representative immunoblots after incubation with Notch (DAPT) and EGFR inhibitors (AG1478) on 1 day after BMP exposure. Inhibitors were incubated same as Fig. 3a. j Relative quantification of P-EGFR and NICD in i (n = 2, 4). Data represent means ± s.e.m. (*P < 0.05, ***P < 0.005, ****P < 0.001, *****P < 0.0001; Student’s t-test for d and f, Tukey’s test for h and j, n.s. not significant). Scale bars, 20  µm. Source data of immunoblots are provided as a Source Data file.