Figure 2

NRF2 expression is upregulated in SCN1A^severe patient iNSCs after H₂O₂ treatment. (a) The expression of NRF2 gene resulted significantly up-regulated in SCN1A^severe patient iNSCs in respect to WT and SCN1A^mild cells at 16 h of 2 mM H₂O₂ treatments. (b) Immunoblot analysis showed that the NRF2 protein increased after 8, 16 and 24 h of H₂O₂ treatment in both WT and SCN1A^severe patient iNSCs, but for each time point tested we observed a higher expression of NRF2 in patient cells compared to WT cells. (c) Bands optical density (OD) of western blot showed in (b), calculated as NRF2 bands OD normalized on GAPDH band OD on the same time point. Data are presented as mean ± SEM of three biological replicates. (d) 8 and 16 h of H₂O₂ treatment induced a prominent NRF2 nuclear translocation in patient iNSCs in respect to SCN1A^mild and healthy cells, as shown by quantification of NRF2 immunofluorescence signal overlapping with the nucleus (representative images are shown in Supplementary Fig. S2, at least five image per condition were analysed); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t-test. (e) Expression of NRF2 target genes NQO1 and HMOX1 after 8, 16 and 24 h of H₂O₂ treatment in WT and patients iNSCs. For qPCR, GAPDH was detected as loading control and data are presented as mean ± SEM of three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t-test.