Chromatin-binding NE proteins collaborate during NE formation. (A) Diagram shows the dynamic localization of nuclear-targeted GFP (green) during open mitosis. Reaccumulation of GFP-NLS into daughter nuclei serves as an indicator for completed NE formation. (B) Cells were transfected with H2B-tdTomato and GFP-NLS and imaged through mitosis. Representative traces of chromatin-localized GFP-NLS in which t = 0 is set at the onset of chromosome separation show the time required for NE formation in U2OS cells with reduction of protein levels by siRNA knockdown. (C) Average time from chromosome separation to GFP-NLS nuclear accumulation was plotted. n > 20 for each condition (Table S1) with P < 0.01 when LBR, Lap2β, MAN1, BAF, Ndc1, or Pom121 siRNA was compared with scrambled (scram) RNA control, and P = 0.23 and 0.20 for Sun1 and Nup107, respectively (by t test). (D) U2OS cells were transfected with H2B-tdTomato (red) and Sec61-GFP (green, black, and white insets) and imaged from mitosis. Nuclear rim formation was compared in cells transfected with scrambled RNA or siRNA against Lap2β (closed arrowheads). After 12 min, no nuclear rim was detected with the knockdown of Lap2β (open arrowheads) compared with rim signal present in scrambled siRNA controls. Outlined areas represent the regions that are magnified below. Bar, 20 µm. (E) Average time from chromosome separation to complete nuclear rim formation was plotted. P < 0.01 when Lap2β, BAF, or Ndc1 knockdown was compared with scrambled RNA. Dotted lines indicate control cell timing. Error bars indicate SEM.