Fig. S5

Fig. S5 CPT induces nuclear envelope blistering and TOP1cc exit to the cytoplasm, related to Figure 5 (A) Representative electron microscopy images for a distinct set of cells. Treatment was conducted for 30 min with 50 nM CPT. Scale bar, 500 nm. Arrows pointing at blister structures. Nu, nucleoplasm; Cy, cytoplasm; A, autophagosome. (B) Representative electron tomographic slices fixed by high-pressure freezing showing nuclear envelope blister in HeLa. Treatment was conducted for 30 min with 50 nM CPT. Scale bar, 2 μm (left); scale bar, 1 μm (zoom, right). Arrows pointing at blister structures. (C) Immunofluorescence of TOP1cc foci and cyclin B1 after 3 h of treatment with 50 nM CPT (n = 3). Cyclin B1 is used as a control for the inhibition of exportin by leptomycin B (LTB). Scale bar, 10 μm. (Left) Quantification of positive cells for cytoplasmic TOP1cc foci. One-way ANOVA. (Right) Cyclin B1 intensity per nuclei (quantification for a representative experiment). t test. Error bar, SD. ∗p < 0.05; ∗∗∗p < 0.0005; ns, not significant. (D) Cryo-focused ion beam (FIB) milling and cryo-fluorescence correlation images of cells after 30 min of 50 nM CPT treatment. Lysosomes are stained with LysoTracker. (Top) Ion beam image of a final lamellae after targeted milling and correlation between the SEM image of a lamellae, the transmission electron microscopy (TEM) medium magnification montage of the same lamellae acquired at 15,000× (white dashed lines) and the fluorescent molecularly imprinted polymer (MIP) acquired post milling. (Bottom) Close-up view on the TEM medium magnification correlated with LysoTracker fluorescence used for targeted acquisition of the tilt series. Scale bar, 2 μm.