Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: NGF-induced PC12 cell differentiation toward sympathetic neurons and expressions of GAP-43 and TH. (A) Immunofluorescent stains of GAP-43 (red, upper row) and TH (red, lower row). Nuclei were stained blue by DAPI. Note that NGF induced significant neurite outgrowth. Scale bar, 10 μm. (B,C) Real-time PCR and western blotting results of GAP-43 and TH expressions in PC12 cells treated or untreated with NGF. ∗∗∗ p < 0.001 vs. respective control. ### p < 0.001 vs. respective control; n = 3 for each value.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Cell Differentiation, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: The suppressing effects of ivabradine (IVA) on the mRNA and protein expressions of HCN channel isoforms and GAP-43 and the neurite outgrowth. (A–D) Real-time PCR and western blotting results of the mRNA and protein expression levels respectively for HCN1-4 isoforms in NGF-treated PC12 cells relative to GADPH level. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. NGF treatment alone in the mRNA level. # p < 0.05, ## p < 0.01 vs. NGF treatment alone in the protein level. (E) Representative western blotting bands ( upper ) and the mRNA and protein semiquantitative values ( lower ) of GAP-43 in NGF-treated PC12 cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. NGF treatment alone in the mRNA level. # p < 0.05, ### p < 0.001 vs. NGF treatment alone in the protein level. (F) Immunofluorescent stains of GAP-43 protein (red), displaying that NGF induced, while ivabradine inhibited, the neurite outgrowth in differentiating PC12 cells. DAPI was used for cell nuclei staining (blue). Scale bar, 10 μm. (G,H) Quantitative morphological parameters of neurite outgrowth including the total neurite length and the maximal neurite length (longest process per cell). Results are calculated from three independent experiments and presented as mean ± SEM, 96 < n < 213, ∗ p < 0.05, ∗∗ p < 0.01 vs. NGF treatment alone.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: 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.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Expressing, Transfection, Negative Control, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron

doi: 10.3389/fncel.2019.00415

Figure Lengend Snippet: 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.

Article Snippet: The membranes were blocked overnight with 5% BSA and then were respectively incubated with polyclonal anti-HCN1 (APC-056), anti-HCN2 (APC-030), anti-HCN3 (APC-057), anti-HCN4 (AGP-004) at 1:1000 dilution (Alomone Labs, Jerusalem, Israel), or with anti-GAP-43 (sc-135915) or anti-TH (sc-136100) at 1:1000 dilution (Santa Cruz Biotechnology, Inc. United States) overnight at 4°C.

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot