Fig. 4: Cell death analysis.
From: HuD impairs neuromuscular junctions and induces apoptosis in human iPSC and Drosophila ALS models
A Representative brightfield images of co-cultures at day 28. Scale bar: 20 μm. B Quantitative analysis of dead cells using ethidium homodimer-1 at day 7, 14, 21, 28 of co-cultures obtained with FUSWT SkMCs and FUSWT, FUSP525L, or FUSWT + HuD MNs. The graph shows the average (dots) and standard deviation from 3 batches of differentiated cells. For each replicate, 5 fields have been acquired at each time point. Ordinary two-way ANOVA: p = 1.2 × 10−9 for genotypes comparison, p = 3.0 × 10−15 time points comparison, p = 2.0 × 10−7 for their interaction; post hoc Tukey test p values for multiple comparisons among genotypes within each time point, and among day 7 and later time points within each genotype, are indicated in the table. See Supplementary Fig. S3 for representative images of the cells. C Representative merged images of immunofluorescence staining of day 14 co-cultures (left panels) or MN monocultures (right panels) using Cleaved Caspase-3 (CC-3) and MAP2 antibodies, and DAPI. Scale bar: 50 μm. Single panels are shown in Supplementary Fig. S4. D, E The graphs report quantitative analysis of the CC-3 fluorescence intensity at day 14 and 28 of MNs monoculture (D) or co-cultures (E). Each dot represents a replicate, consisting of an individual batch of differentiated iPSCs. For each replicate, 6 fields have been acquired and the dot shows the average value. Error bars indicate standard deviation calculated on the average value of the replicates. Ordinary two-way ANOVA. No significant differences resulted from the comparison of MN monocultures (D). For the co-cultures (E), p = 2.1 × 10−7 for genotypes comparison, p = 0.0003 for time points comparison, p = 0.0324 for their interaction; relevant post hoc Tukey test p values for multiple comparisons are indicated in the graph. Source data are provided as a Source Data file.
