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. 2022 Apr 12;4:846669. doi: 10.3389/fgeed.2022.846669

FIGURE 3.

FIGURE 3

CRISPR-mediated activation of Itgav supports complex neurite arborization of differentiated N2a cells. (A) Time course of the experiment. (B) Representative images of N2a cells expressing the indicated constructs. Transfection was verified by EGFP expression, β-tubulin III staining was used to trace neurites and Hoechst to stain nuclei. (C) Percentage of cells with neurites within EGFP-positive cells for experiments as in (A,B). **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey’s post-test (n = 6 coverslips from 5 independent experiments). CRISPRa for either Itgav or Itgb3 or both doubles the percentage of differentiated N2a cells, as compared to control conditions. (D–F) Average (D), longest (E) and total neurite length (F) of differentiated N2a cells expressing the indicated constructs. *p < 0.05, ***p < 0.001, one-way ANOVA followed by Tukey’s post-test (n = 6 coverslips from 5 independent experiments). (G) Morphological classification of differentiated N2a cells. *p < 0.05, ***p < 0.001, Chi-square test (n = 55, 82, 75, and 83 cells from 5 independent experiments for gRNA Ctrl, gRNAs Itgav, gRNAs Itgb3 and gRNAs Itgav + Itgb3, respectively). (H) Sholl analysis of differentiated N2a cells. *p < 0.05, **p < 0.01 relative to gRNA Ctrl, repeated measures ANOVA followed by Dunnett’s post-test (n = 55, 82, 75, and 83 cells from 5 independent experiments for gRNA Ctrl, gRNAs Itgav, gRNAs Itgb3, and gRNAs Itgav + Itgb3, respectively). CRISPRa for Itgav induces a complex arborization.