Lamin B loss in nuclear blebs is rupture dependent whereas increased DNA damage is rupture independent.

The nucleus must maintain shape and integrity to protect the function of the genome. Nuclear blebs are deformations identified by decreased DNA density that commonly lead to rupture. Lamin B levels often vary drastically between blebs. We tracked rupture via time-lapse imaging of nuclear localization sequence (NLS)-GFP immediately followed by immunofluorescence imaging of lamins and known rupture markers. We find that lamin B1 loss consistently marks ruptured nuclear blebs better than lamin A/C, emerin and cGAS. Visualizing post-rupture lamin B1 loss and emerin enrichment reveals that cell lines display widely different propensities for nuclear bleb rupture. To determine how rupture affects DNA damage, we time-lapse-imaged ruptured and unruptured blebs, then conducted immunofluorescence on the same cells for DNA damage markers γH2AX and 53BP1. We find that DNA damage is increased in blebbed nuclei independently of rupture. This was verified in blebbed LNCaP nuclei, which do not rupture and maintain lamin B1, but still show increased DNA damage. Thus, we confirm that lamin B is the most consistent marker of nuclear rupture, and that blebbed nuclei have increased DNA damage regardless of rupture.