Fig. 5

JNK signaling activation and D-JNKI1’s protective effects in Rett human iPSCs. a Neuronal differentiation of human iPSCs. The upper panel showed the neuronal differentiation protocol. The timing of critical steps was indicated in days from day 0 (d0). At the end of the differentiation (red arrow, d40), neurons were exposed to 2μM D-JNKI1 for 48 h before neurons were isolated for Western blot analysis. Immunofluorescence was used to define cell identity (lower panel): a1- OCT4/SSEA4 staining for iPSCs, a2- Nestin and SOX1 staining for telencephalic neural progenitors (d12/13), and a3- β3-Tubulin (TuJI) staining for neurons in terminally differentiated cultures and DAPI to stain nuclei. Scale bar: iPSCs and NPCs 100 μm and neurons 20 μm. b Western blots and quantifications of JNK activation in neurons differentiated from hiPSCs from RTT human patients; clones expressing either the wild type (hMecp2^wt) or the mutated (hMecp2^mut) MECP2 allele were differentiated in neurons. The hMecp2^mut displayed higher P-JNK/JNK and P-c-Jun/c-Jun ratios than to hMecp2^wt neurons. D-JNKI1 reduced hMecp2^mut activation to hMecp2^wt levels; D-JNKI1 in hMecp2^wt neurons did not change P-JNK/JNK (n=4 and 5) and P-c-Jun/c-Jun ratios (n=5). c Cell death in hMecp2^wt and hMecp2^mut neurons: the hMecp2^mut showed greater cell death than to hMecp2^wt. D-JNKI1 reduced induced-cell death in the hMecp2^mut neurons to the control level (hMecp2^wt). Data were shown as mean ± SEM. Significance was calculated using two‐way ANOVA followed by Bonferroni post hoc test (panel b). Significance vs control *p<0.05, **p<0.01, and ****p<0.0001; D-JNKI1-treated vs untreated mutated neurons #p<0.05, ##p<0.01, and ###p<0.001. See additional file S3