Validation of LA- and LC-KD and the effect of LC-KD on the leakage of NLS-Halo from the nucleus to the cytoplasm. (A and B) Validation of LA- and LC-KD with immunofluorescence (A) and immunoblotting (B). (A) Representative immunofluorescence images of single confocal sections in WT MEFs expressing scrambled control, shLA or shLC with sfCherry stained with anti-LA (4A58, left panel) and anti-LC (ab125679, right panel), followed with Cy5-labeled anti-mouse and rabbit IgG, respectively, and Hoechst 33342 for DNA. Bar: 20 μm. (B) Whole cell lysates from WT MEFs expressing the indicated shRNAs were probed with anti-LA/C and anti-GAPDH (as loading control). Positions of the size standards are shown on the right. (C and D) During time-lapse imaging of WT MEFs expressing scrambled control (shScr) or shLC with 1 min intervals, a 2-μm diameter spot was laser-microirradiated to induce NE rupture (yellow arrowheads). (C) Dynamics of NLS-Halo in response to NE rupture in the indicated cells. The right four columns are magnified views of orange boxes. Bars: 5 μm (the first column) and 2 μm (the second to fifth column). (D) The cytoplasmic-to-nuclear intensity (C/N) ratio of NLS-Halo was measured and plotted to monitor NE rupture (means ± SEM; n = 10 cells; *, P < 0.05 from control by a Mann-Whitney U test). Source data are available for this figure: SourceData FS2.