Fig. 4

FOS expression in CREB-deficient cortical neurons after KCl treatment. a Cultured cortical neurons were transfected with either pTα1-EGFP alone (control) or the CRISPR/Cas9 vector together with pTα1-EGFP (CRISPR/Cas9). The cultures were fixed at 7 DIV after 2 h KCl treatment. ICC was performed with multi-color fluorescence labeling of EGFP (Alexa488), CREB (Cy3), FOS (Cy5, far red) and DAPI. Scale bar 10 μm. b Distribution histogram shows the CREB expression level in the CRISPR/Cas9-transfected neurons (black) and the control neurons (white). The distribution of the CRISPR/Cas9-transfected neurons is significantly different from the control neurons (KS test, P < 0.001). An interrupted vertical line indicates a boundary between the estimated fraction of the homozygous mutant neurons and others. c Scatter diagram shows the expression of FOS and CREB in the neurons transfected with CRISPR/Cas9 vector and pTα1-EGFP (CRISPR/Cas9, black) and pTα1-EGFP transfected neurons (control, white) at 7 DIV. An interrupted vertical line indicates a boundary between the estimated fraction of the homozygous mutant neurons and others. d Dot plots show the FOS expression level in the CRISPR/Cas9-transfected neurons (control, white) and the CRISPR/Cas9-resistant rescue Creb cDNA and the CRISPR/Cas9-cotransfected neurons (black). The number of neurons analyzed in each case is presented in parentheses. Bars represent the mean. Asterisks indicate a significant difference from control cells (Mann–Whitney’s U test, **P < 0.01)