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. Author manuscript; available in PMC: 2024 Jul 4.
Published in final edited form as: Nat Aging. 2024 Jan 4;4(1):62–79. doi: 10.1038/s43587-023-00549-0

Fig. 2: Downregulation of age-dependent genes drives NSPC aging.

Fig. 2:

a, Heatmap of down-regulated and up-regulated genes in aged versus young NSPCs. Values of log2(FPKM) from three replicates of RNA-seq data are plotted. b, Gene ontology analysis of the down-regulated and up-regulated genes as in (a). Two-sided Hypergeometric test. c, Dot plot showing relative cell viability of NSPCs (siRNA group vs. siControl group) 48 hours after siRNA transfection. n = 3 independent experiments. d, Heatmap of representative siRNAs that significantly decrease the expression of target genes and the cell viability of NSPCs. The relative expression of genes was analyzed by qPCR from three independent experiments (siRNA group vs. siControl group, P < 0.05, p-values are provided in the source data). e, Quantification of neurospheres formed by single NSPCs 48 hours post-transfection of siRNAs as indicated. n = 4 independent experiments for siControl group. n = 3 independent experiments for siRNA group. f, EdU incorporation assay with young NSPC-derived neurospheres 48 hours post-transfection of siRNAs. Data are plotted with the percentage of EdU-positive cells versus Hoechst-positive cells in each neurosphere. siControl n = 45, siBarhl2–1 n =37, siBarhl2–2 n = 31, siDmrta2–1 n = 47, siDmrta2–3 n = 35, siHlx-2 n = 38, siHlx-3 n = 33, siIgf2bp2–1 n = 38, siIgf2bp2–3 n = 45, siIgf2bp3–2 n = 41, siIgf2bp3–3 n = 47, siLix1–1 n = 37, siLix1–2 n = 31, siNkain2–2 n = 32, siNkain2–3 n = 31, siTmem28–1 n = 47, siTmem28–3 n = 44, siTrf-1 n = 39, siTrf-2 n = 34, siTwist1–2 n = 34, siTwist1–3 n = 35. g, Representative images of EdU labeling of NSPC-derived neurospheres as in (f). Hoechst (blue) marks nuclei. Scale bar, 100 μm. h, Western blot of IGF2BP2 protein in the gene knockdown and rescue groups. n = 3 independent experiments. i, Representative images of neurospheres treated with knockdown of Igf2bp2 with and without expression rescue of Igf2bp2. Control of rescue was infected with adenovirus without expressing Igf2bp2. EdU labeling of neurospheres was performed 48 hours post-transfection. DAPI (blue) marks nuclei. Scale bar, 100 μm. Quantification data are showed in the bar chart with the percentage of EdU-positive versus DAPI-positive cells in each neurosphere. n > 35. Error bars are mean ± SD for e, f, and i. Two-sided Student’s t-tests for c-f, and i.