Fig. 4

RACK1 knockdown alleviates aggregation and global translational suppression by TDP-43^ΔNLS and WT over-expression in HEK293T cells. a Representative SUnSET-Western Blot (WB) demonstrates that compared to control, expression of HA-tagged TDP-43^ΔNLS and to a lesser extent over-expression of WT TDP-43, induces a significant reduction in puromycin (PMY) incorporation. b RACK1 siRNA KD alleviates global translational levels in both cases as determined by quantification of PMY band intensities normalized to loading control β-actin. c SUnSET-ICC shows inhibition of PMY incorporation by TDP-43^ΔNLS (HA) expression preferably occurs in cells containing distinctive cytoplasmic aggregates (asterisks, top row). In cells where TDP-43^ΔNLS displays a filamentary expression pattern (arrows, bottom row), PMY incorporation is comparable to neighbouring HA negative UT cells. d RACK1 KD alleviates aggregation, resulting in diffuse cytoplasmic expression (top row) or predominantly nuclear localization (bottom row) of TDP-43^ΔNLS in a sub-population of transfected cells and correlating with normal PMY incorporation compared to neighbouring HA-negative UT cells. Nuclei stained with DAPI in merged images. Statistics: Ordinary one-way ANOVA Tukey multiple comparisons. n = 4 *p < 0.05; ***p < 0.001; ****p < 0.0001. Scale bars: 20 μm