TREx microscopy reveals lamin B1 network and NE deformations induced by nuclear polyQ aggregates. (A) Depth color coded (left panel) and maximum intensity projection (right panel) of a representative expanded (TREx) U2OS cell with cytosolic polyQ74 aggregates. (B) Zooms of various cytosolic aggregates shown in A. (C–E) Images of representative expanded U2OS cell immunolabeled for lamin B1 (magenta) and counterstained with DAPI (gray). (C) Orthogonal view showing discrete localization of lamin B1 at the nuclear periphery. (D) Sum projection of the top 1.5 μm of the nucleus shown in C, showing a zoom of the lamin B1 meshwork. (E) 3D render of the whole lamin B1 meshwork of cell shown in C. Cropped render of the middle of the nucleus reveals nucleoplasmic reticulum present in U2OS cells. (F and G) Representative images of expanded U2OS cells expressing polyQ74-NLS (F) or polyQ74 (G) stained for lamin B1. Lamin B1 intensity profile indicates disrupted (F) or intact (G) lamin B1 meshwork at aggregate. (H) Control U2OS cell stained with mCLING (magenta) showing a distinct NE (orange arrow). (I) Images of expanded U2OS cell expressing polyQ74-NLS and HA-mCherry-cGAS (cyan), showing NE deformation and invagination around a nuclear aggregate, at a NE rupture location marked by cGAS accumulation. (J) Intensity profile of NE rupture site shown in G showing mCLING stained NE accumulation around a polyQ fibril. (K) Representative images of expanded neurons expressing cytosolic polyQ74 aggregates (orange) labeled using a total protein stain (NHS; grayscale). Scale bars indicate 2.5 μm for overviews and 0.5 μm for zooms (A–D, F–J, and K). Voxel sizes are 2 μm (grids in E).