Skip to main content
. 2019 Sep 20;13:415. doi: 10.3389/fncel.2019.00415

FIGURE 4.

FIGURE 4

Knocking-down HCN2 and HCN4 expression with siRNAs inhibited GAP-43 expression and neurite outgrowth in PC12 cells. NGF-treated PC12 cells were transiently transfected with the negative control siRNA (NC-siRNA) or with the siRNA respectively for HCN1, HCN2, HCN3 or HCN4 using Lipofectamine® RNAiMAX Reagent for 48 h before cells were harvested for detection. (A–D) The mRNA and protein expressions of HCN1-4 isoforms assayed by qPCR and western blotting in comparison to GADPH in NGF-treated PC12 cells. ∗∗p < 0.01, ∗∗∗p < 0.001 vs. negative control siRNA (N.C.) in the mRNA level. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. N.C. in the protein level. (E) Representative western blotting results (upper) and the densitometric analysis of GAP-43 mRNA and protein expressions (lower) (n = 3–6 independent experiments). ∗p < 0.05, ∗∗∗p < 0.001 vs. N.C. in the mRNA level. ###p < 0.001 vs. N.C. in the protein level. (F) Immunofluorescent stains of GAP-43 in PC12 cells transiently transfected with siRNA targeting HCN2 or HCN4. Scale bar, 10 μm. (G,H) Quantification of neurite morphological parameters (total length of neurite outgrowth and longest process per cell). Results are calculated from three independent experiments and presented as mean ± SEM, 50 < n < 84, ∗p < 0.05, ∗∗p < 0.01 vs. N.C.