Quantification of NE/ER membrane removal from chromatin. (A) Experimental setup to monitor removal of NE/ER membranes from chromatin. HeLa cells stably expressing the INM protein GFP-LAP2β as an NE/ER marker, H2B-mRFP as a chromatin marker, and mPlum-GST-M9 as a nuclear efflux marker defining the onset of NEBD (t = 0 min) were used for live-cell confocal microscopy. Image acquisition of early prophase cells was performed every 2 min in four z slices. (B) Representative images of reporter cells before and after NEBD. Note that the strong emission of H2B-mRFP is also visible in the mPlum channel. To detect efflux of mPlum-GST-M9 into the cytoplasm, all images of the mPlum channel were processed in parallel by enhancing levels followed by setting γ correction to 0.5. (C) Removal of GFP-LAP2β–positive membranes from chromatin was determined using an ImageJ-based routine that measures the distance between the innermost NE/ER membrane and the outermost chromatin signal along 360 lines (blue lines; enlarged in boxed area) of a radial grid emanating from the center of mass of chromatin. (D) Reporter cells in early prophase were treated with DMSO or 333 nM nocodazole, cytochalasin D, or latrunculin B and imaged by confocal microscopy as described (A). Representative images show progression of membrane removal from chromatin at selected time points before and after NEBD. (E) Line blots show quantitative analysis comparing treatment with the different inhibitors (six experiments; means ± SEM; n ≥ 15 per condition). Bars, 5 µm.