Fig. 5

NCDN depletion in neurons affects FUS cytoplasmic granule dynamics. Lentivirus containing shRNAs targeting NCDN (NCDN-KD1 or -KD2) or non-targeted scramble (CTL-KD) were used to deplete primary rat cortical neurons (RCN) of NCDN. a Confocal images of RCN (DIV16) stained with antibodies against FUS (HPA008784, green), MAP2 (red) and DAPI (blue). Scale bar = 10 μm. b IMARIS generated 3D surface images of FUS-positive cytoplasmic granules from RCN. Quantification of the number of granules per neuron (c), mean area (d) and volume of granules (e). f Bootstrapped difference of maximum intensity of segmented FUS cytoplasmic granules from NCDN-KD compared to CTL-KD RCN showing the kernel density plot of the bootstrapped differences (shaded grey area), the minimum and maximum resampled differences (black horizontal lines), and the 95% confidence interval (red horizontal lines). NCDN-KD1,95% CI: 0.0062- 0.1937 p = 0.0098; NCDN-KD2,95% CI: 0.0044–0.1372 p < 0.0001. g Western blot of NCDN, FUS and GAPDH proteins from RCN. h Quantification of FUS protein levels from RCN relative to GAPDH. Statistical analysis was performed using a one-way ANOVA with multiple comparisons using Turkey test (c–e) or a Student’s t test (h) (a, p < 0.05; b, p < 0.01; c, p < 0.005; d, p < 0.001 vs CTL; ns, not significant, p > 0.05 vs CTL). Error bars represent the mean ± SEM. Each experiment was performed from n = 3–4 biological replicates per group