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. 2007 Jul 23;27(19):6706–6717. doi: 10.1128/MCB.01225-07

FIG. 4.

FIG. 4.

Shp2 is required for NSC proliferation and self-renewal in vitro. (A) Morphological examination shows the reduced number and size of Shp2 mutant neurospheres as compared to controls. Scale bar, 100 μm. (B) Neurosphere assay. NSCs were initially seeded at 5 cells/μl, and the number of neurospheres (NS) was determined after 7 days for primary (1°), secondary (2°), and tertiary (3°) neurospheres (**, P < 0.01; n = 3). 1k, 1,000. (C) Generation of secondary neurospheres. The frequency was expressed as the percentage of secondary neurospheres generated versus the total number of single cells dissociated from primary neurospheres. Self-renewal capacity was defined as the number of secondary neurospheres formed per primary neurosphere (*, P < 0.05; **, P < 0.01; n = 4). (D) Growth curve of NSCs in self-renewal culture. Neurospheres in suspension were gently dissociated into single cells and then enumerated (**, P < 0.01; n = 3). K, 1,000. (E) Growth curve of NSCs in differentiation culture. Single NSCs were seeded on a precoated plate in the absence of bFGF and EGF to induce neuronal differentiation; 2% FBS was added after 24 h to induce astroglial differentiation. Cell proliferation was monitored using the RT-CES system (17). (F) BrdU incorporation assay in NSCs (Nestin+), neurons (TuJ1+ or MAP2+), and astrocytes (GFAP+). (Left panel) Representative pictures of BrdU+ cells in NSCs. (Right panel) Statistical analysis. Scale bar, 50 μm. NSCs: mutant, 18.1% ± 7.5%; control, 54.4% ± 11.2%; **, P < 0.01; n = 5. Neurons: mutant, 15.3% ± 6.1%; control, 32.5% ± 7.1%; *, P < 0.05; n = 3. Astrocytes: mutant, 32.3% ± 4.2%; control, 43% ± 12%; P > 0.05; n = 3). (G) Cell apoptosis (TUNEL) assay in NSCs (Nestin+), neurons (TuJ1+ or MAP2+), and astrocytes (GFAP+). (Left panel) Representative pictures of apoptotic cells in NSCs. Scale bar, 100 μm. (Right panel) Statistical analysis. NSCs: mutant, 12.2% ± 7.2%; control, 8.9% ± 4.6%; P > 0.05; n = 5. Neurons: mutant, 13.7% ± 4%; control, 9.2% ± 1; *, P > 0.05; n = 3. Astrocytes: mutant, 8.3% ± 0.3%; control, 8.9% ± 1%; P > 0.05; n = 3.