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. 2019 Jul 22;30(16):1985–1999. doi: 10.1091/mbc.E19-02-0090

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© 2019 Smith et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 6: — Constricted migration of human cells induces nuclear damage and MyoD loss. (A) Representative images of nonmigrated and migrated human MuSCs (hMuSCs) on the top and bottom, respectively, of a 3 μm–pore transwell membrane. White arrow indicates a nuclear bleb formed after 3 μm–pore migration. Scale bar = 20 μm; inset scale bar = 5 μm. (B) Migration of hMuSCs through 3-μm pores causes a significant increase in nuclear blebbing. N = 22–37 cells per condition from three experiments. (C) Constricted migration of hMuSCs also leads to a significant increase in DNA damage, as measured by γH2AX foci. (D) (i) Nuclear bleb frequency is elevated among Rh30 cells that migrate through 3-μm pores vs. cells that migrate through 8-μm pores or cells that remain unmigrated on top of the transwell membrane. (ii) Images show a representative Rh30 nucleus with a migration-induced bleb. N = 88–142 cells. Scale bar = 10 μm. (E) Migration of Rh30 cells through 3-μm pores causes excess DNA damage. N = 88–142 cells. (F) Representative images of Rh30 cells after migration through 3- or 8-μm pores. Line scan through two nuclei (a, b) after migration through 3-μm pores. Scale bar = 10 μm. (G) Normalized intensity based on the line scan in F. Cell (a) shows high coincident DNA and MyoD signal and an absence of cGAS. Cell (b) shows cGAS signal overlapping with DNA and low nuclear MyoD intensity. Cell (b) appears to have ruptured, causing nuclear entry of cGAS and mislocalization of MyoD from the nucleus into the cytoplasm. (H) The frequency of MyoD-negative Rh30 nuclei—like cell b—increases significantly following 3 μm–pore migration. N = 314–370 cells. (I) (i) Addition of blebbistatin to both sides of a 3-μm transwell greatly reduces migration. (ii) Bar graph: Rh30 cells expressing DNA repair protein GFP-KU80 and DNA-binding protein mCherry-cGAS migrated through 3-µm pores; DNA repair factor KU80 mislocalizes to cytoplasm at bottom, except with blebbistatin. Inset: cells showing GFP-KU80 mislocalized to cytoplasm in Ctl bottom also exhibit mCherry-cGAS accumulation in the nuclear bleb. (J) After migration through 3-μm transwell pores, cells show mislocalization of GFP-tagged and immunostained Ku80, accompanied by focal mCherry-cGas indicative of nuclear rupture. Blebbistatin prevents such nucleocytoplasmic exchange (17–93 cells on the bottom; >300 cells on the top). * indicates significant (p < 0.05) difference from no migration.