channel expression in iPSC-derived neurons. (A) Real-time PCR addressing neuronal Nav expression at 30 days of differentiation (N = 3 in each cell line) Crossing point differences to β-actin (ΔCp = Cpβ-actin - CpNav) closer to zero indicate higher expression. PCR efficiencies were nearly identical (Additional file 2). Asterisks indicate a significant difference to SCN1A (P < 0.5, one-way ANOVA). Expression strength of the indicated Nav genes was constant across the cell lines (P = 0.92, two-way ANOVA) (B) Normalized expression levels for each Nav gene (SCN1A + SCN2A + SCN3A + SCN8A)/4 = 1. Compared to the control, SCN1A expression tended to be higher in D1-1 (P = 0.0929, one-way ANOVA), and it was significantly higher in D1-6 (*P = 0.0078). The distribution of Nav genes expression ratios in each cell line was significantly different between the control and the patient lines (P =0.0086 and <0.0001 for D1-1 and D1-6, respectively, two-way ANOVA), but identical between D1-1 and D1-6 (P = 0.11). (C) Sequencing of SCN1A reverse transcribed mRNA isolated from iPSCs-derived neurons. Patient-neurons show a double peak at mutation site (solid arrowheads), confirming the heterozygous state of the cells (D) Immunocytochemical characterization of Nav1.1 expression in control neurons: strong (solid arrows), moderate (open arrows), weak (solid arrowheads), and faint (open arrowhead). Despite weak staining in the cell body, neurite staining was often apparent (solid arrowheads). (E) Neurite co-localization of Nav1.1 and the AIS marker ankyrin G (AnkG, solid arrowheads). (F) PAN-Nav staining of SCN1A Venus-positive neurons (via anti-GFP, see Figure 3) in the AIS (arrowheads). (G) Co-localization of Nav1.1 and GAD67 staining. (H) VGlut1-positive neuron with SCN1A Venus expression. Scale bars: 100 μm (D), 30 μm (F), 200 μm (G) and 50 μm (others).