Depletion of cyclin B induces pre-NEBD slippage. (A) Abnormal mitosis with intact lamin A coupled with cytoskeleton rearrangement in cyclin B-depleted cells. ^mAIDB1^KOB1B2 cells were synchronized using a double thymidine block and released into a drug-free or DI-containing medium. After 12 h, cells were fixed and imaged using Airyscan confocal microscopy. Representative images show untreated cells in interphase or mitosis and a DI-treated cell undergoing aberrant mitosis. Scale bar: 5 µm. (B) Depletion of cyclin B leads to mitosis devoid of NEBD. ^mAIDB1^KOB1B2 cells were transfected with plasmids expressing EGFP-BAF and histone H2B-EGFP. After 36 h, the cells were synchronized using a single thymidine block and released into either a drug-free or DI-containing medium. After 10 h, live-cell imaging was performed using Airyscan confocal microscopy. Representative images of a control cell entering mitosis (note the DNA condensation and the redistribution of EGFP-BAF) and a cyclin B-depleted cell lacking breakdown of EGFP-BAF-containing nuclear lamina are shown. Cell outlines are indicated by white dotted lines. Time: h:min. Scale bar: 10 µm. (C) Silencing of cyclin B results in mitosis without loss of nuclear membrane integrity. ^mAIDB1^KOB1B2 cells were transfected with an RFP-NLS expression plasmid, cultured in drug-free or DI-containing medium, and analyzed using live-cell imaging. Representative images show normal mitosis and aberrant mitosis without NEBD. Time: h:min. Scale bar: 10 µm. The graph represents the cumulative percentage of cells that have progressed past NEBD, as judged by the flooding of RFP-NLS signal (n = 50). (D) Absence of NEBD in cyclin B-depleted cells. ^mAIDB1^KOB1B2 cells were transfected with a plasmid expressing mRFP-lamin A and cultured in a drug-free or DI-containing medium. Representative images from live-cell imaging analysis of normal mitosis and abnormal mitosis are shown (NEBD denoted by an asterisk). Time: h:min. Scale bar: 10 µm. See Videos 3 and 4.