Figure 4. Cytoplasmic poly-GA aggregates inhibit nuclear import of p65.

(A) Cytoplasmic poly-GA aggregates inhibit p65 nuclear translocation. HEK293 cells were transfected with empty vector (Control), NES-GA₆₅-GFP, NLS-GA₆₅-GFP, GA₆₅-GFP, GA65-GFP-PY, or Htt96Q-GFP (Htt96Q) (green) and analyzed for NF-κB p65 localization (red) with and without TNFα treatment (30 min). White dashed lines delineate nuclei based on DAPI staining. Scale bar represents 10 µm. (B) Quantification of NF-κB p65 distribution from data in (A). The x-axis shows the enrichment of p65 in the cytoplasm relative to the nucleus. Data are means + SD (n = 3), >100 cells were analyzed per condition. **p≤0.01, ***p≤0.001 from two-sided t-test. (C) Expression of poly-GA does not alter the degradation of IκB and phosphorylation of p65. HEK293 cells were transfected with the indicated constructs (Ct: Control; NES: NES-GA₆₅-GFP; NLS: NLS-GA₆₅-GFP; NT: GA₆₅-GFP; PY: GA₆₅-GFP-PY) and treated as described in (A). Levels of IκB and phosphorylated NF-κB p65 (P–p65) were analyzed by immunoblotting. α-tubulin served as loading control.

Figure 4—figure supplement 1. Importins form aggregates in cells containing cytoplasmic poly-GA aggregates and are partially sequestered.

HEK293 cells were left untreated (Control) or were transfected with GA₆₅-GFP, NES-GA₆₅-GFP or NLS-GA₆₅-GFP (green). Cells were then analyzed by immunofluorescence with antibodies against importin α-1 (KPNA2; A), importin α-3 (KPNA4; B), and importin β-1 (KPNB1; C) (red). Close-up views of inclusions are shown, and arrowheads indicate importin aggregates and/or their sequestration at the periphery of the inclusion. The co-localization profile along the white arrow is shown, plotted as percentage of maximal fluorescence on the y-axis. The white dashed lines delineate nuclei based on DAPI staining. Scale bars are 10 µm.