Complete rescue of DNA damage and cell cycle defect requires antioxidant and MYO-i, revealing a bimodal dependence of cell cycle on DNA damage. (A) Reactive oxygen species (ROS) damage DNA but can be neutralized by reduced glutathione, including a membrane-permeable ethyl ester (GSH-MEE, denoted GSH in the figure). Constricted migration increases ROS based on intensity of MitoSOX dye (>50 cells per condition, n = 3 experiments; *, P < 0.05). Bot., bottom. (B) GSH-MEE partially rescues the excess DNA damage after constricted migration. Complete rescue of DNA damage requires both MYO-i or KUsBR and GSH-MEE on both sides of the Transwell membrane, but drugs on bottom suffice (>80 cells per condition, n = 3 experiments; *, P < 0.05). All scale bars: 10 µm. Ctl, control. (C) GSH-MEE alone does not affect constricted migration or nuclear rupture (more than three fields of view, n = 3 experiments; *, P < 0.05). n.s., not significant. (D) Both control and GSH-MEE show 4N suppression after constricted migration, but the cell cycle block is rescued by GSH-MEE+MYO-i or KUsBR even with MYO-i only on bottom (>80 cells per condition, n = 3 experiments; *, P < 0.05). Pop., population. (E) For a pore size–dependent test of cell cycle suppression, we enlarged commercial pores by NaOH etching to novel diameters of 2.20, 2.30, 3.85, 3.95, and 5.0 µm. The plot shows that the percentage of 4N remains suppressed on bottom unless DNA damage is fully rescued. The Hill equation, y = A + B^m/[(x − 1)^m + C^m], has fit parameters (m = 15, A = 0.67, B = 0.22, C = 0.23, R² = 0.78) and goes through error bars for 13 of 15 data points. Data from pore migration rescue experiments (expts.; Figs. 1, 2, and 3) are also fitted. Representative error bars (8 µm and control) for pore and pipette experiments are shown (>50 cells per condition, n = 3 experiments).