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. 2025 Aug 12;36(9):ar111. doi: 10.1091/mbc.E25-02-0057

FIGURE 6:

FIGURE 6:

LAP2β and LB1 codepletion increases lamin A/C meshwork defects in an actin-dependent manner. (A) Relative transcript abundance of lamins and various INM proteins in DLD1 cells; Nucleoplasmic TMPO splice isoform LAP2α distinguished from INM-localized splice isoform LAP2β by isoform-resolved analysis with Salmon (see Materials and Methods). (B) Overview of experimental strategy. mNG-AID-LB1 cells were subject to RNAi for 48 h followed by 16 h of auxin treatment. (C–F) Immunofluorescence of lamin A/C, mNG-AID-LB1, and actin (phalloidin) in mNG-AID-LB1 cells after control RNAi (C and D) or LAP2β RNAi (E and F) along with vehicle (C and E) or auxin (D and F). All conditions received 1 h vehicle treatment as control for G–K. (G) Normalized histograms of the percentage of lamin A/C-free nuclear surface area in mNG-AID-LB1 cells after 48 h control RNAi treatment plus 16 h auxin treatment (blue) or 48 h LAP2b RNAi treatment plus 16 h auxin treatment (orange). Curves indicate mean and error bars indicate SD of three independent experiments. (H) Summary plot of the percentage of nuclei with contiguous lamin A/C (<1% lamin A/C-free nuclear surface, gray), with gaps in the lamin A/C meshwork covering >1% of the nuclear surface (orange), or with gaps covering >10% of the nuclear surface (red) in mNG-AID-LB1 cells in the indicated conditions. ****, p < 0.0001 by the Fisher's exact test. (I) Overview of experimental strategy. mNG-AID-LB1 cells were subject to RNAi for 48 h followed by 16 h of auxin treatment, then 1 h of Lat A treatment. (J–M) Immunofluorescence of lamin A/C, mNG-AID-LB1, and actin (phalloidin) in mNG-AID-LB1 cells after control RNAi (J and K) or LAP2β RNAi (L and M) along with vehicle (J and L) or auxin (K and M). All conditions received 1 h Lat A treatment; scale bar, 10 µm. (N) Quantification of the percentage of nuclei with lamin A/C defects detected by blinded categorical scoring. Each dot on graph represents one experimental replicate; n > 400 cells analyzed per condition across at least three replicate experiments. ns indicates p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001 by the unpaired t test.