Studying NE rupture and viability in cells expressing polyQ aggregates. (A) U2OS-RFP-NLS (magenta) cells expressing polyQ74-NLS aggregates (green). (B) Timelapse of RFP-NLS signal (inverted grayscale) of cell shown in A showing multiple blebbing events (black arrowheads) and NE rupture evidenced by cytosolic leaking of RFP-NLS signal (black arrow). (C) Graph showing nuclear (magenta curve) and cytosolic (black curve) RFP-intensity, indicating multiple instances of changes in nuclear and cytosolic RFP-NLS signal (green arrows 1, 2, and 3). After the last nuclear rupture (green arrow 4), nuclear enrichment does not restore. (D) Percentages of cells showing various amounts of nuclear ruptures during 8-h imaging. (E and F) Time-lapse images of representative U2OS-RFP-NLS cells (magenta and inverted grayscale) and polyQ74-NLS (green) showing blebbing event (orange arrowheads) without NE rupture. (G) Graph of nuclear RFP-intensity of timelapse in F, showing no loss of RFP-NLS signal during blebbing event. (H and I) Time-lapse images of representative U2OS-RFP-NLS cells expressing cytosolic polyQ74 aggregates (green), showing a characteristic bean shaped nucleus (black arrow) and NE deformation near a smaller cytoplasmic aggregate (black arrowhead). (J) Nuclear RFP-intensity of timelapse in I showing no loss of RFP-NLS signal during imaging. (K and L) Representative images of live-cell imaging of U2OS-RFP-NLS cells expressing polyQ74-NLS (green) that were labeled using Mitoview (grayscale) showing maintenance (K) or loss (L) of mitochondrial membrane potential in cells with unhealed NE ruptures. (M) Quantification of the fraction of cells with unhealed NE ruptures showing maintenance, or loss, of mitochondrial membrane potential (n = 80; N = 3). All scale bars indicate 10 μm, except for F and I and zooms of B (5 μm).