Journal: Cell reports
Article Title: SoxD genes are required for adult neural stem cell activation
doi: 10.1016/j.celrep.2022.110313
Figure Lengend Snippet: (A) Confocal images showing immunostaining for YFP, BrdU, and GFAP of control and Sox5 icKO mice brain sections at P90 at 30 dpi. Yellow arrows indicate recombined RGL cells that retain BrdU after 1 month (YFP + BrdU + rGFAP + cells). (B) Experimental design for BrdU label retention experiment. (C) Quantification of BrdU + cells among YFP + recombined cells and BrdU + rGFAP + YFP + cells among rGFAP + YFP + cells. Each symbol represents an independent biological replicate. (D) Scheme of adult NSC preparation grown as neurospheres. (E) Immunohistochemistry in hippocampal neurospheres for Sox2 and Sox5. (F) Neurospheres dissociated and seeded as attached cells and cultured with FGF2 alone or in combination with BMP4. A 40 min BrdU pulse was given before fixation, and BrdU incorporation was analyzed using immunohistochemistry. (G) Quantitation of Sox5 protein levels in NSCs in FGF2 and in FGF2 plus BMP4 conditions expressed in arbitrary units relative to the FGF2 condition. p = 0.0001 by Student’s t test (n = 300 cells). (H) qPCR analysis for the indicated transcripts in NSCs grown with FGF2 plus BMP4 with respect to those grown with FGF2 alone. Results are shown as 2 −ΔΔCt normalized with respect to two housekeeping genes ( Pgk1 and Gapdh ) and relative to values in cells grown in FGF2 alone (dashed line on y axis = 1). A total of three or four independent neurospheres cultures were used, and three technical replicates were performed. (I) Immunohistochemistry of nucleofected NSCs (YFP + cells; green), using either pCIG or pCIG-Sox5 vectors, to analyze proliferating Ki67 + cells in each condition. (J) Quantitation of the percentage of Ki67 + cells in each indicated condition. In all graphs, data are mean value ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired (C) or paired (H and J) Student’s t test. Scale bars represent 50 μm (A), 30 μm (E), and 80 μm (F and I).
Article Snippet: To induce quiescence, cells were plated at a density of 80,000 cells/MW24 well in basal media with 20 ng/ml FGF2 and after 24 h, media was replaced with basal media with 20 ng/ml FGF2 alone or in combination with with 30 ng/mL recombinant mouse BMP4 (100 ng/ μl; PeproTech).
Techniques: Immunostaining, Control, Immunohistochemistry, Cell Culture, BrdU Incorporation Assay, Quantitation Assay
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