Figure 1. MYO-i on bottom rescues nuclear rupture and DNA damage but not cell cycle suppression. (A) Nuclei rupture in constricted migration through Transwells of customized pore size that also allow asymmetric exposure to drugs. (B) Time-lapse images of A549 cell expressing GFP-lamin-A as nonphosphorylatable S22A and emerging from a 3-µm pore, with a bleb (arrows) forming at the leading tip of the nucleus. GFP-lamin-A accumulates in the bleb independently of S22 phosphorylation (see also Fig. S1 B). (Ci) In constricted migration of U2OS cells, nuclear blebs form with lamina disruptions (arrows) except when blebbistatin (MYO-i) is on bottom (Bot.). Inset: Rupture occurs occasionally (arrow) with MYO-i. (Cii) Addition of MYO-i to the 3-µm bottom or both sides of a Transwell greatly reduces migration and nuclear (Nucl.) blebs but increases circularity (Circ.). The 8-µm Transwell is used as a control (ctl; >100 cells per condition, n ≥ 3 experiments *, Pxy < 0.05; Pxy is the joint probability obtained by multiplying px and py, which assumes independence; Wang, 2013). (D) U2OS cells expressing DNA repair protein GFP-KU70 and cytoplasmic DNA-binding protein mCherry-cGAS migrated through 3-µm pores; most cells showed GFP-KU70 mislocalized to cytoplasm, and mCherry-cGAS accumulated in the nuclear bleb in control (Ctl). MYO-i prevents such nucleocytoplasmic exchange (n = 3 experiments). Bar graph: Endogenous DNA repair factor KU80 also mislocalizes to cytoplasm (cyto.), except with MYO-i or with larger pores that eliminate blebs (50∼300 cells, n > 3 experiments, *, P < 0.05). Dist., distance; Rel. Int., relative intensity. (E) DNA breaks constantly form and are repaired, but if net DNA damage is high, then damage checkpoints block cell cycle progression. Phosph’n denotes phosphorylation. (F) Foci of γH2AX (white in image) are not enriched in nuclear blebs (arrows) after 3-µm pore migration. Bar graphs: γH2AX foci measured in confocal projections are in excess on bottom except with MYO-i or with larger pores. Compared with the nuclear body, blebs are low in lamin-B as expected but equal in foci density (>100 cells, n = 5 experiments; *, P < 0.05). n.s., not significant. (G) Using EdU spike-in to label replicating DNA during Transwell migration, DNA stain intensity and EdU were used to identify a cell as 2N (nonreplicated genome) or 4N (fully replicated genome) and as G1, early S (eS), late S (lS), or G2 (see Fig. S1 G). When contact-inhibited cells migrate through large (8-µm) pores into sparse microenvironments, cells reenter cell cycle. Constricting (3-µm) pores block cell cycle and suppress mitosis (Mito.), regardless of MYO-i. No significant difference is seen between 2N/4N populations on bottom or top after treatment with blebbistatin for both 3- and 8-µm pores (n.s.; >400 cells per condition, n = 3 experiments; *, P < 0.05). All scale bars: 10 µm.
Figure 1.

MYO-i on bottom rescues nuclear rupture and DNA damage but not cell cycle suppression. (A) Nuclei rupture in constricted migration through Transwells of customized pore size that also allow asymmetric exposure to drugs. (B) Time-lapse images of A549 cell expressing GFP-lamin-A as nonphosphorylatable S22A and emerging from a 3-µm pore, with a bleb (arrows) forming at the leading tip of the nucleus. GFP-lamin-A accumulates in the bleb independently of S22 phosphorylation (see also Fig. S1 B). (Ci) In constricted migration of U2OS cells, nuclear blebs form with lamina disruptions (arrows) except when blebbistatin (MYO-i) is on bottom (Bot.). Inset: Rupture occurs occasionally (arrow) with MYO-i. (Cii) Addition of MYO-i to the 3-µm bottom or both sides of a Transwell greatly reduces migration and nuclear (Nucl.) blebs but increases circularity (Circ.). The 8-µm Transwell is used as a control (ctl; >100 cells per condition, n ≥ 3 experiments *, Pxy < 0.05; Pxy is the joint probability obtained by multiplying px and py, which assumes independence; Wang, 2013). (D) U2OS cells expressing DNA repair protein GFP-KU70 and cytoplasmic DNA-binding protein mCherry-cGAS migrated through 3-µm pores; most cells showed GFP-KU70 mislocalized to cytoplasm, and mCherry-cGAS accumulated in the nuclear bleb in control (Ctl). MYO-i prevents such nucleocytoplasmic exchange (n = 3 experiments). Bar graph: Endogenous DNA repair factor KU80 also mislocalizes to cytoplasm (cyto.), except with MYO-i or with larger pores that eliminate blebs (50∼300 cells, n > 3 experiments, *, P < 0.05). Dist., distance; Rel. Int., relative intensity. (E) DNA breaks constantly form and are repaired, but if net DNA damage is high, then damage checkpoints block cell cycle progression. Phosph’n denotes phosphorylation. (F) Foci of γH2AX (white in image) are not enriched in nuclear blebs (arrows) after 3-µm pore migration. Bar graphs: γH2AX foci measured in confocal projections are in excess on bottom except with MYO-i or with larger pores. Compared with the nuclear body, blebs are low in lamin-B as expected but equal in foci density (>100 cells, n = 5 experiments; *, P < 0.05). n.s., not significant. (G) Using EdU spike-in to label replicating DNA during Transwell migration, DNA stain intensity and EdU were used to identify a cell as 2N (nonreplicated genome) or 4N (fully replicated genome) and as G1, early S (eS), late S (lS), or G2 (see Fig. S1 G). When contact-inhibited cells migrate through large (8-µm) pores into sparse microenvironments, cells reenter cell cycle. Constricting (3-µm) pores block cell cycle and suppress mitosis (Mito.), regardless of MYO-i. No significant difference is seen between 2N/4N populations on bottom or top after treatment with blebbistatin for both 3- and 8-µm pores (n.s.; >400 cells per condition, n = 3 experiments; *, P < 0.05). All scale bars: 10 µm.

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