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. 2019 Sep 20;13:415. doi: 10.3389/fncel.2019.00415

FIGURE 5.

FIGURE 5

Overexpression of HCN2 and HCN4 enhanced GAP-43 expression and neurite outgrowth in PC12 cells. Results were obtained 48 h after cell transfection with either empty plasmid pcDNA3.0/vector or pcDNA3.0/HCN2 or pcDNA3.0/HCN4. (A,B) Representative western blots (upper) and semiquantitative values (lower) of the mRNA and protein levels of HCN2 and HCN4 isoforms. ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control (pcDNA3.0/vector) in the mRNA level. #p < 0.05 vs. control (pcDNA3.0/vector) in the protein level, n = 4−6 independent experiments. (C) qPCR and western blotting results showing the effects of HCN2 or HCN4 overexpression on the mRNA and protein expressions of GAP-43. ∗∗∗p < 0.001 vs. empty plasmid in the mRNA level. ###p < 0.001 vs. empty plasmid in the protein level, n = 4−6 independent experiments. (D) Immunofluorescent stains of GAP-43 (red) of PC12 cells transiently transfected respectively with pcDNA3.0/vector, pcDNA3.0/HCN2 or pcDNA3.0/HCN4 plasmids. Scale bar, 10 μm. (E,F) Quantification of neurite morphological parameters including total neurite outgrowth and longest process per cell. Results are calculated from three independent experiments and presented as mean ± SEM, 50 < n < 90, ∗p < 0.05 vs. empty vector.