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. 2015 Aug 4;36(8):966–975. doi: 10.1038/aps.2015.48

Figure 5.

Figure 5

Omi activity and E2F1 level are correlated in regulation of neurite outgrowth. (A) A neurite outgrowth assay was performed showing that UCF-101, an Omi activity inhibitor, inhibits neurite outgrowth in N2a cells. UCF-101 was added to N2a cells at a final concentration of 20 μmol/L for 30 min before RA treatment for another 48 h. The bar graph shows the average neurite length of each group measured by Image J software (bar, 20 μm; number of cells, approximately 200; bP<0.05, one-way ANOVA). (B) A neurite outgrowth assay was performed, showing that knocking down Omi by sh-Omi containing a GFP sequence inhibited neurite outgrowth in N2a cells and knocking down E2F1 in sh-Omi-transfected cells restored neurite outgrowth. Scale bar, 20 μm; (C) The bar graph shows the average neurite length of each group measured by Image J software (number of GFP-positive cells, approximately 200; bP<0.05, one-way ANOVA). (D) The effect of si-E2F1 was proved by detecting the protein level of E2F1. N2a cells were transfected with a control siRNA (NC) or siRNA that targets E2F1 (si-E2F1) for 72 h. Whole-cell extracts were prepared and immunoblotted with the E2F1 antibody. (E) The mRNA of E2F1-targeted gene cyclin E1 was upregulated in sh-Omi transfected N2a cells, and this effect was reversed by knocking down E2F1. qRT-PCR assays were performed using primers specific for cyclin E1 and β-actin genes. n=3 per group. bP<0.05; one-way ANOVA.