Fig 1: Expression of SCN1A gene and NaV1.1 protein in idNs. (A) Sequencing of cDNA obtained by reverse transcription of SCN1A mRNA from WT- and SCN1AM145T-idNs. In the SCN1AM145T cells, the double peak in the mutation site (indicated by the orange arrow) demonstrates that both alleles (one with the original nucleotide T and the other with the mutated one C) were transcribed. (B) qRT-PCR analysis of SCN1A gene in idNs at day of differentiation 0 (NSCs), d14, d21 and d28. SCN1AM145T cells showed a lower expression of SCN1A during differentiation compared to WT, although in both cell lines the expression increases following idNs maturation. (C) The expression of the embryonic isoform of sodium channel SCN3A increases with the progress of cell maturation in SCN1AM145T idNs, while it decreases in WT idNs as the cells become more differentiated. For both graphs, GAPDH was used as a housekeeping gene; data are mean ± SEM of three biological replicates (black dots), * p < 0.05, ** p < 0.01, t-test has been calculated vs. WT at the same day of differentiation. (D) Western blot analysis of NaV1.1 protein in lysates obtained from WT- and SCN1AM145T idNs at day 35 of differentiation. Tubulin Beta 3 Class III (TUBB3) was used as loading control. (E) Quantification of NaV1.1 Western blot bands in four biological replicates (n = 4, OD = relative optical density calculated as (NaV1.1 optical density)/(TUBB3 optical density), p-value calculated using t-test). (F) Immunofluorescence of idNs showing that GABAergic neurons (GAD1 positive) express NaV1.1, mainly in the cell body. Immunofluorescence data show a lower expression of the channel in the neurons differentiated from SCN1AM145T patient in respect to those of WT (63× magnification).