Extended Data Fig. 5: The rejuvenating effects of age-dependent genes on aged NSPCs.
a, Quantification of SOX2-positive cells in the SVZ. The average percentage of SOX2+ /DAPI+ cells was plotted based on data from three aged wild-type mice. A total of eight sections per mouse, spaced at 120 μm intervals along the rostrocaudal axis of the SVZ, were used for cell counting. Cells were quantified within the lateral and dorsolateral SVZ in each section. b–d, Quantification of BrdU+SOX2+ NSPCs in the left (Igf2bp2, Igf2bp3, and Barhl2 overexpression) and right (wild-type) sides of the SVZ. The adenovirus encoding these genes were injected in the left side of SVZ of aged mice, respectively. BrdU labeling was performed two weeks after the gene transduction. BrdU+SOX2+ NSPCs were quantified at both sides of the lateral and dorsolateral SVZ in each section. Seven sections per mouse. Three mice per group. Unpaired two-sided Student’s t-test. (b): P = 0.0396, (c): P = 5.05E-09, (d): P = 1.39E-08. * P < 0.05. **** P < 0.001. e, f, Overexpression of Igf2bp2 and Igf2bp3 increases the number of DCX-positive cells in the SVZ of aged mice. Control mice were injected with control adenovirus without expressing these genes. Confocal microscopy images showing immunofluorescence co-labeling of DCX and DAPI in the SVZ. Scale bar, 20 μm. DCX-positive cells were quantified at the injection site within the lateral and dorsolateral SVZ in each section. A total of six sections per mouse, spaced at 120 μm intervals along the rostrocaudal axis of the SVZ, were used for cell counting. Three mice per group. The average number of DCX-positive cells per group was plotted blow the images. Unpaired two-sided Student’s t-test. (e): P = 0.0355, (f): P = 0.0216. * P < 0.05. g–i, Quantitative real-time PCR analysis of relative expression of Igf2bp2, Igf2bp3 and Barhl2 40 hours after the adenovirus-mediated transduction of the respective genes in primary aged NSPCs. The control group was transduced with control adenovirus without expressing these genes. n = 3 independent assays. Unpaired two-sided Student’s t-test. j, Cell death analysis by propidium iodide (PI) staining in primary aged NSPCs transduced with the genes of Igf2bp2, Igf2bp3 and Barhl2. NSPC-formed neurospheres were dissociated into single cells for staining 48 hours following adenovirus-mediated transduction of the respective genes. Scale bar, 200 μm. k, Quantification data of PI staining shows the percentage of PI-positive cells among Hoechst-positive cells, which were counted from 10 microscopic fields per group. Error bars are mean ± SD for b-k.
