Chapter 6 194 Pol III protein expression is disease status dependent Western blot analysis showed a significant difference in Pol IIIA protein expression between cell types (χ2(2) = 11.252, p = 0.004). Post-hoc pairwise comparisons revealed decreased Pol IIIA protein expression in neurons compared to iPSCs (p = 0.0152) and in neurons compared to NES (p = 0.035, Fig. 2A1). Additionally, we identified a significant decrease in 4H cells compared to control cells (Fig. 2A2; χ2(1) = 4.857, p = 0.028). Similarly, analysis of Pol IIIB protein expression was performed, for this subunit there was no significant difference based on cell subtypes (χ2(2) = 4.812, p = 0.090; Fig. 2B1 ). Though, expression of Pol IIIB subunit was again significantly lower in 4H patient cells compared to controls (χ2(1) = 6.204, p = 0.013; Fig. 2B2) In conclusion, 4H status affects Pol III protein levels, suggesting that 4H may alter Pol III subunit expression (independent of cell type). Minimal impact of individual differences on Pol III protein levels To understand the contribution of individual differences to protein expression of Pol IIIA and Pol IIIB, we again calculated the ICC. We identified minimal influence of individual differences on Pol III protein expression, with only 6.1% of residual variance for Pol IIIA and 0% for Pol IIIB being explained by inter-individual variability. So unlike gene expression, Pol III protein levels are not substantially affected by individual genetic differences. ▶ Figure 2: Pol III protein subunit A and B expression in different cell types. Relative protein expression of Pol III subunit A (A) and Pol III subunit B (B) as determined by western blot, comparing cell type (iPSC, NES and neurons) and control against 4H. Open circles represent controls, solid circles represent individuals with 4H caused by POLR3A (dark) or POLR3B (light) variants. Disease status graphs (A2, B2) represent the average relative protein expression across all three cell types, indicated with squares. Horizontal lines across circles/squares reflect the median. Data was analyzed using a linear mixed-effects model after removal of influential outliers (indicated with “). * Significance was assessed at p < 0.05, ** p < 0.01 or *** p < 0.001.
RkJQdWJsaXNoZXIy MTk4NDMw