Fig. 1
From: A human iPSC-derived inducible neuronal model of Niemann-Pick disease, type C1
Generation of an NPC1 mutant i3Neuron cell line. A The genomic structure of NPC1, reference DNA sequence, and targeting guide RNA are shown in the top part of this panel. Sanger sequencing of NPC1−/− i3Neuron clones identified two independent deletions in exon 4 of NPC1. Allele 1 is a 5-bp exonic deletion (c.437-441del p.Tyr146fsX167), and allele 2 is a 64-bp deletion (c.434_463+36del) extending from exon 4 to intron 4. A minimum of 4 clones corresponding to each allele were sequenced, and no other NPC1 mutations were detected. B Protein levels of NPC1 and β-actin in NPC1+/+ and NPC1−/− i3Neurons were analyzed using Western blotting. Blots are representative of three independent experiments. C NPC1+/+ and NPC1−/− i3Neurons were differentiated for 10 days, and endolysosomal accumulation of unesterified cholesterol was visualized by staining with PFO-488 (green) and anti-LAMP1 (red). The nuclei were counter-stained with Hoechst (blue). Data are from 30 cells per condition in average: representative of 5 independent experiments (n = 5). Lamp1 area of puncta counts was calculated from n ≥ 10 cells and averaged per cell. Scale bar, 10 μm. Magnified inset scale bar, 1 μm. D Lipid levels measured in NPC1+/+ and NPC1−/− i3Neurons by LC-MS. Data is plotted relative to NPC1+/+ i3Neuron levels. Dotted line represents an NPC1−/−/NPC1+/+ ratio of 1. Individual points represent independent measurements, and one independent experiment is a representative of three samples for each group. E NPC1+/+ and NPC1−/− i3Neurons were stained with anti-GM2 ganglioside (green) and Hoechst nuclear stain (blue) 10 days post-differentiation. Data are from at least 500 cells per condition, representative of 3 independent experiments (n = 3). Scale bar, 10 μm. *p < 0.05, **p < 0.01, ****p < 0.0001 by Mann-Whitney test when comparing two independent samples