Figure 3
Effect of cell density over IDS-ko NSCs differentiation. (a–f) Coimmunostaining for caspase3+ and ubiquitin with the neural differentiation markers β-tubIII, GFAP and GalC of wt and IDS-ko NSCs at 0 and 17 div of differentiation under HD (1.25 × 104 cells/cm2) and LD (6.5 × 103 cells/cm2) cell density conditions. (a) Confocal microscopy images showing the coimmunostaining of caspase3 marker with GFAP, GalC or β-tubIII under HD or low LD conditions. Scale bar: 100 μm. (b) Quantification of GFAP+, β-tubIII+ and GalC+ cells over total DAPI+ nuclei under HD and LD conditions. (c) Quantification of caspase3+ cells over total DAPI+ nuclei under HD and LD conditions. (d) Quantification of GFAP+/caspase3+, β-tubIII/caspase3+, GalC+/caspase3+ cells over total caspase3+ cells under HD and LD conditions. (e) Confocal microscopy images showing the immunostaining of ubiquitin+ marker. Scale bar: 100 μm. (f) Quantification of ubiquitin+ cells over total DAPI+ nuclei under HD and LD conditions. (g) Confocal microscopy images showing the presence of caspase3+ cells in wt (upper left) and IDS-ko (upper right) mouse cortex. Scale bar: 75 μm; in inset: 11.72 μm. Quantification of caspase3+ cells over total DAPI+ nuclei (bottom left) and colocalization with glial markers GFAP (bottom center) and MBP (bottom right). Scale bars: 21, 10 μm. (h) Confocal microscopy images showing the presence of ubiquitin+ cells in wt (upper left) and IDS-ko (upper right) mouse cortex. Scale bar: 75 μm. Inset scale bar: 16 μm. Quantification of ubiquitin+ cells over total DAPI+ nuclei (bottom left) and colocalization with GFAP (bottom center) and MBP (bottom right). Scale bar: 13.7 μm. (i) Confocal microscopy images showing the presence of caspase3+ cells in wt (upper left) and Hunter (upper right) human cortex (end stage). Scale bar: 75 μm. Quantification of caspase3+ cells over total DAPI+ nuclei (bottom left) and colocalization with GFAP (bottom center), MBP (bottom right) and neuronal marker β-tubIII (bottom right). Scale bar: 10–14 μm. (j) Confocal microscopy images showing the presence of ubiquitin+ aggregates in wt (upper left) and Hunter (upper right) human cortex (end stage). Scale bar: 75 μm. Quantification of caspase3+ cells over total DAPI+ nuclei (bottom left) and colocalization with GFAP (bottom center), MBP (bottom right) and β-tubIII (bottom right). Scale bars: 16–18 μm. Student’s t-test was applied, the differences among all the values were statistically not significant unless indicated (*P≤0.05,**P≤0.01, ***P≤0.001) values are means±S.E.M. Casp, active caspase 3; Ub, ubiquitin
