Figure 4.

Reduced morphological complexity of cells with decreased expression of Pol III subunits. (A) Representative immunofluorescence images (from four independent experiments with similar results) of siRNA-treated OLs in differentiation conditions, showing MOG+ cells (green). Nuclei stained with DAPI. Scale bar, 20 μm (20X). (B) Representative examples of OL processes from control or Pol III siRNA-treated cells as schematized diagrams. (C) Categorization of OL morphological maturity, as determined by branching complexity and myelin-like sheath formation, of MOG+ cells collected after 3DIV in differentiation media. (D) Schematic of Sholl analysis, in which a series of concentric rings are super-imposed on the cell as a measure of process networks (parameters - inner radius 20 μM, outer radius 140 μM, step size 10uM). (E) Quantification of Sholl analysis, depicted as mean ± SEM number of intersections at each ring from four independent experiments (each presented as an average of 5–8 technical replicates). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired, two-sided Student’s t-test. (F,G) Quantification of (F) field area and (G) total length analysis in siRNA-treated cells labelled with MOG. (H) Representative immunofluorescence images (from three independent experiments with similar results) of siRNA-treated OLs in differentiation conditions, showing O4+ cells (red). Nuclei stained with DAPI. Scale bar, 20 μm (20X). (I,J) Quantification of (I) field area and (J) total length analysis in siRNA-treated cells labelled with O4. (K) Representative immunofluorescence images (from three independent experiments with similar results) of siRNA-treated OLs in differentiation conditions, showing MOG+ cells (green) immunolabelled for CC3 (red). Nuclei stained with DAPI. Scale bar, 20 μm (20X). (L) Percent cell death as determined by ReadyProbe assay. Data presented as fold change of control condition graphed as mean ± SEM.