Skip to main content

Advertisement

Fig. 3 | Molecular Brain

Fig. 3

From: Proteasome impairment in neural cells derived from HMSN-P patient iPSCs

Fig. 3

Gene correction of TFG P285L mutation and improvement of cellular phenotypes in HMSN-P. a Gene targeting strategy used to generate mutation-corrected iPSC clone. CRISPR-Cas9 targeting the TFG locus created a double strand break upstream of exon 8. Homologous recombination of the genomic locus with a targeting plasmid with control sequence of exon 8 coupled puroΔTK replaced the TFG P285L mutant allele. After puromycin selection, the resistance cassette was removed by transposon expression and FIAU selection. DSB: double strand break, FIAU: 1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil. b Sequencing of exon 8 of TFG in mutation-corrected iPSCs showed the correction of the TFG P285L mutation. c Immunostaining for TFG in purified MNs by HB9::GFP sorting. Scale bar = 10 μm. d Quantification of area of TFG-positive puncta in purified MNs by HB9::GFP sorting measured by high-content analysis (n = 9, **p < 0.01, one-way ANOVA followed by Tukey’s multiple comparison post hoc test). Error bars are ± s.e.m. e Immunoblot analysis of TFG in control, HMSN-P patient, and mutation-corrected iPS-MNs. β-actin was used as loading control. f Proteasome activity in control, HMSN-P patient, and mutation-corrected iPS-MNs (n = 3, * p < 0.05, one-way ANOVA followed by Tukey’s multiple comparison post hoc test). Error bars are ± s.e.m. g Survival of control, HMSN-P patient, and mutation-corrected iPS-MNs after MG132 exposure (n = 3, **p < 0.01, one-way ANOVA followed by Tukey’s multiple comparison post hoc test). Error bars are ± s.e.m. h Immunoblot analysis of cleaved caspase-3 in control, HMSN-P patient, and mutation-corrected iPS-MNs after exposure of vehicle or MG132. DMSO: dimethyl sulfoxide

Back to article page