Figure 3. Excess DNA damage in cells... | Rockefeller University Press

$Figure 3. Excess DNA damage in cells correlates with mislocalized DNA repair factors. (A) i: Ruptured A549 shLMNA nuclei show high cytoplasmic KU80 by immunofluorescence; the lamina is focally enriched in lamin A (arrow) and depleted in lamin B. ii: 92% of rupture events occur at poles of nuclei where curvature is high. Cartoon shows 2D curvature in cells. iii: Ruptured nuclei have lower circularity, indicating high curvature. Overall nuclear area is the same. n > 150 cells in three experiments. (B) Lamin A knockdown increases the fraction of cells with mislocalized cytoplasmic KU80 and excess DNA damage as indicated by γH2AX foci. GFP-LMNA rescues both effects. γH2AX foci in nuclei of shLMNA cells are not enriched near lamina rupture sites (arrows). n > 150 cells in three experiments. (C) For shLMNA cells, ruptured nuclei with higher cytoplasmic/nuclear KU80 have higher γH2AX foci counts compared with nonruptured ones with low cytoplasmic/nuclear KU80. Ctl and GFP-LMNA–rescued cells rarely rupture and show low cytoplasmic/nuclear KU80 and low γH2AX foci counts. n > 150 cells in three experiments. *, P < 0.05. (D) Time-lapse imaging: Mislocalized cytoplasmic GFP-53BP1 recovers over hours into nuclei. Shape fluctuations of U2OS siLMNA nuclei create high-curvature regions (arrow) before second GFP-53BP1 mislocalization. Cytoplasmic/nuclear GFP is plotted over time (n = 5 cells). Right plots: Nuclear circularity and area over time. High curvature occurs <1 h before rupture. Bars, 10 µm. All data are present as mean ± SEM.$

Figure 3.

Excess DNA damage in cells correlates with mislocalized DNA repair factors. (A) i: Ruptured A549 shLMNA nuclei show high cytoplasmic KU80 by immunofluorescence; the lamina is focally enriched in lamin A (arrow) and depleted in lamin B. ii: 92% of rupture events occur at poles of nuclei where curvature is high. Cartoon shows 2D curvature in cells. iii: Ruptured nuclei have lower circularity, indicating high curvature. Overall nuclear area is the same. n > 150 cells in three experiments. (B) Lamin A knockdown increases the fraction of cells with mislocalized cytoplasmic KU80 and excess DNA damage as indicated by γH2AX foci. GFP-LMNA rescues both effects. γH2AX foci in nuclei of shLMNA cells are not enriched near lamina rupture sites (arrows). n > 150 cells in three experiments. (C) For shLMNA cells, ruptured nuclei with higher cytoplasmic/nuclear KU80 have higher γH2AX foci counts compared with nonruptured ones with low cytoplasmic/nuclear KU80. Ctl and GFP-LMNA–rescued cells rarely rupture and show low cytoplasmic/nuclear KU80 and low γH2AX foci counts. n > 150 cells in three experiments. *, P < 0.05. (D) Time-lapse imaging: Mislocalized cytoplasmic GFP-53BP1 recovers over hours into nuclei. Shape fluctuations of U2OS siLMNA nuclei create high-curvature regions (arrow) before second GFP-53BP1 mislocalization. Cytoplasmic/nuclear GFP is plotted over time (n = 5 cells). Right plots: Nuclear circularity and area over time. High curvature occurs <1 h before rupture. Bars, 10 µm. All data are present as mean ± SEM.

This Feature Is Available To Subscribers Only