Figure 5.

Remodelin reverses prelamin A‐dependent defects in aged VSMCs. (A) IF of p11, p30 and p30 +Remodelin VSMCs. Cells were stained for γH2AX, NUP153 and 53BP1 (all green) in addition to prelamin A (red) and DAPI (blue). Panels showing 53BP1 staining show cells at a reduced magnification to allow visualization of staining in cell cytoplasmic regions. (B) Quantification of average number of γH2AX in p11, p30 and p30 + Remodelin VSMCs. n > 100 cells were counted per cell group from 3 independent experiments. Standard errors are shown. (C) Nuclear circularity of p11, p30 and p30 + Remodelin VSMCs. Values closer to 1 indicate nuclei that are more circular. n > 100 cells from more than 3 independent experiments. (D) Fluorescence intensity measurements of cytoplasmic 53BP1 in p30 and p30 + Remodelin VSMCs. Readings for nuclear and cytoplasmic were obtained, and percentage cytoplasmic values were calculated. n > 100 cells from 3 independent experiments. (E) WB of whole cell and cell fractions (cytoplasmic – C, nuclear – N) of p30 VSMCs −/+ Remodelin (R). (F) Quantification of 53BP1 bands shown in D. Data are from 3 separate experiments. (G) Quantification of γH2AX bands shown in D. Data are from 3 separate experiments (H) (Left) Co‐immunoprecipitation WB using NUP153 as bait with lamin AC and prelamin A as target interactors. Assays were performed in U2OS cells expressing UCLA −/+FTIs and −/+ Remodelin. Prelamin A acted as a competitor against lamin AC to bind to NUP153, but this was alleviated to an extent by addition of Remodelin. (Right) Quantification of lamin A and C precipitation. Band intensities of precipitated lamin AC were measured and normalized to total input lamin AC. Fold changes of precipitated lamin AC in UCLA, UCLA + FTI and UCLA + Remodelin were then calculated relative to EGFP control cells. n = 4, standard errors are shown.