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. 2012 Jun 6;32(23):7926–7940. doi: 10.1523/JNEUROSCI.4303-11.2012

Figure 5.

Figure 5.

Notch 1 signaling regulates self-renewal of Rad-NSCs. A, NICD1 (green) was expressed in tdRFP-positive Rad-NSCs (red) in culture. B, GSI treatment reduced the number of Rad-NSCs compared with DMSO treatment. C, GLAST-positive cells were sorted from mouse brains by MACS. GLAST-positive cells were enriched with GFAP compared with GLAST-negative cells (as determined by immunoblotting). β-Actin levels were used as an internal loading control. D, Following TM treatment of GFAP-CreERTM;Presenilin 1 cKO mice (GP mice), Presenilin 1 expression levels decreased compared with levels in oil-treated GP mice. GAPDH was used as an internal loading control. E, TM treatment of GP mice significantly reduced the number of neural spheres formed in culture compared with control groups. F (CyQUANT assay), G (MTS assay): The number (F) and growth (G) of Rad-NSCs from GP mice were both significantly reduced following 4-OH-TM treatment compared with vehicle control. H, Following TM treatment of GFAP-CreERTM;Notch 1 cKO mice (GN mice), NICD1 expression levels in sorted GLAST-positive cells were decreased compared with levels in control oil-treated mice. I, J, TM treatment of GN mice (I), but not oil treatment (J) removed NICD1 (green) expression in GFAP-positive reactive astrocytes (red). K–M, The numbers of Ki67 (green)-positive proliferating reactive astrocytes (GFAP, red) were compared in oil- or TM- treated GN mice and TM-treated GN heterozygous (hetero) mice. N, TM treatment of GN mice and TM treatment of GN heterozygous mice significantly reduced the number of proliferating reactive astrocytes compared with oil treatment of GN mice. TM treatment of GN mice significantly reduced the number of proliferating reactive astrocytes compared with TM treatment of GN heterozygous mice. O, TM treatment of GN mice and TM treatment of GN heterozygous mice significantly reduced the number of neural spheres formed in culture compared with oil treatment of GN mice. P, Q, 4-OH-TM treatment significantly reduced the proliferation of Rad-NSCs from GN mice and GN heterozygous mice compared with vehicle control (ETOH). P, CyQUANT assay. Q, MTS assay. R, mRNA levels for proteins expressed downstream of Notch signaling were decreased in Rad-NSC spheres derived from TM-treated GN mice compared with oil-treated GN mice. Cell nuclei are shown by DAPI staining (blue). TM, tamoxifen; O, oil. Arrows indicate colocalizations. Scale bars: 50 μm. *p < 0.05; **p < 0.01; ***p < 0.001.