Fig. 3

Transient treatment with Nutlin-3 does not have a persistent effect on intrinsic properties of adult neural stem/progenitor cells. a Experimental scheme for analyzing proliferation and differentiation of hippocampal NSPCs isolated from Fmr1 KO and WT mice treated with Nutlin-3 (Nut3) or vehicle (Veh). b Sample images of proliferating NSPCs pulse-labeled with thymidine analog, BrdU followed by immunohistology for in vitro quantification assay. Red, BrdU; blue, DAPI; scale bar, 20 μm. c Sample images of differentiating NSPCs assessed by immunohistological detection of a neuronal marker Tuj1⁺ for in vitro quantification of NSPC neuronal differentiation. Red, Tuj1; blue, DAPI; scale bar, 20 μm. d, e Nutlin-3 treatment did not rescue impaired proliferation (d) and neuronal differentiation (e) of hippocampal NSPCs isolated from Fmr1 KO and WT mice 4 months after injection (n = 3). f, g Western blot analysis of P-MDM2 levels in isolated NSPCs isolated from Fmr1 KO and WT 4 months after Nutlin-3 or vehicle treatment (n = 3). h–k Western blot analysis of the total histone H3 (T-H3), acetylated histone H3 (acetyl-H3), and HDAC1 levels in NSPCs isolated from Fmr1 KO and WT mice 4 months after Nutlin-3 or vehicle treatment (n = 3). l, m Western blot analysis of P53 levels in isolated NSPCs isolated from Fmr1 KO and WT 4 months after Nutlin-3 or vehicle treatment (n = 3). n, o Western blot analysis of EP300 levels in isolated NSPCs isolated from Fmr1 KO and WT 4 months after Nutlin-3 or vehicle treatment (n = 3). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading controls. Due to the large size of EP300, loading controls were run on a separate gel. *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as means ± SEM