Fig. 5
Analysis of GABAAR subunits, chloride channel expression, and BDNF signaling confirm neuronal immaturity in SEV lines. (A) Ratios of GABRA3 to GABRA1 and (B) GABRA3 to GABRB2 are significantly higher in SEV neurons, indicating a more immature neuronal state compared to HC and MILD lines. Data are presented as mean ± S.E.M. of FPKM values, with each biological replicate representing the ratio of the indicated subunits. *p < 0.05, ** p < 0.01, t-test. (C) Expression of chloride channel genes SLC12A2 (NKCC1) and (D) SLC12A5 (KCC2) in neurons during differentiation. Data are presented as mean ± S.E.M., with each dot representing a biological replicate. (E) The NKCC1/KCC2 ratio is elevated in SEV neurons at both differentiation stages compared to HC and MILD lines. **p < 0.01, ***p < 0.001, t-test. (F) Representative immunostaining of neurons at the late stage of differentiation, showing KCC2 (red) and TUBB3 (green). Insets display a higher magnification, highlighting the localization of KCC2 puncta. (G) Quantification of KCC2-positive puncta per cell in HC, SEV, and MILD neurons at the late stage of differentiation. *p < 0.05, t-test. (H) EGR4 expression during differentiation in HC, SEV, and MILD neurons. (I) Negative enrichment of the Neurotrophin Signaling Pathway in SEV versus MILD comparisons at the late stage of differentiation. (J) Expression of the BDNF gene in HC, SEV, and MILD neurons at both early and late stages of differentiation. (K) Expression of NTRK2 gene (coding for the BDNF receptor) in HC, SEV, and MILD neurons at early and late stages of differentiation.
