Fig. 6: Cul3 haploinsufficiency leads to altered neuron growth, network activity, and to RhoA upregulation in Cul3+/− mice.
a Representative images of 14DIV primary cortical neurons from WT and Cul3+/− mice, immunostained with MAP2 (upper panel), and tracings by simple neurite tracer (lower panel). Scale bar is 25 µm. b–d Quantification of total dendrite length, neurite number, and soma size are shown. Symbols represent independent neurons and color represents littermates. Data are shown as mean ± SD (n = 6 per genotype, at least 6–8 neurons per mouse). Significance is calculated using two-tailed t-test; ***p < 0.001; **p < 0.01. e Representative raster plots of spontaneous spike activity from 8DIV primary cortical neurons. f Spontaneous spike activity is significantly reduced in Cul3+/− cortical neurons; **p < 0.01, two-tailed t-test. g Average burst frequency is significantly reduced in Cul3+/− neurons; **p < 0.01; two-tailed t-test, n = 6–7 mice per genotype; 1.5 × 106 neurons were seeded from each mouse in each MEA plate well, each containing 64 electrodes. Each dot represents independent mouse and color represents littermates. h Representative images of western blot analysis of Cul3, total RhoA, and GAPDH loading control in embryonic and adult cortices. Densitometry analysis of western blot is shown at the bottom. Data are presented as mean ± SEM (n = 6 per genotype). i Representative images of western blot analysis of active RhoA (RhoA-GTP) pulldown, total RhoA and GAPDH loading control from input lysate of embryonic cortex. Densitometry analysis of western blot is shown at the bottom. Data are presented as mean ± SEM (n = 4 per genotype for total RhoA, n = 4 per genotype for active RhoA). Significance is calculated using two-tailed t-test; **p < 0.01. The significance above bars represents comparison against WT.
