Movie S8.

Nuclear envelope rupture during NETosis in human PMN and dHL-60 cells. Human (Hm) PMN were stained with far-red SiR-DNA (Red, right column) and ER-Tracker Red vital dye (green, bottom left) and differentiated neutrophil-like (dHL-60) cells were stained with far-red SiR-DNA (Red, right column) and transfected with either the inner nuclear envelope protein Lap2β fused to mEmerald (green, upper right) or the KDEL sequence combined with the ER-retention signal sequence from calreticulin fused to mEmerald (ER-mEmerald, green, middle right). Time-lapse DIC and spinning-disk confocal images were taken at one min (for Lap2β-mEmerald and ER-mEmerald) or two min (for ER-tracker dye) at 3 μm above the coverslip surface (Z = +3 μm). Cells were stimulated with 4 μM ionomycin at the timepoint when the labels appear. Arrowheads highlight rupture initiation points. Elapsed time shown in min relative to plasma membrane vesiculation. Scale bars = 5 μm (top and middle row) or 10 μm (bottom row).