Fig 2: Nuclear structural defects following Cof/ADF depletion correlate specifically with excessive actomyosin assembly.(a) Confocal immunofluorescence images of control siRNA treated RPE-1 cells with or without expression of constitutively active mDia1 (mDiaCA), labeled for F-actin and LMNA. Second and third columns are single confocal sections at mid nuclear height. Green arrowhead shows perinuclear F-actin accumulation. (b) Quantification of RPE-1 cells with irregular increased numbers of F-actin bundles or the presence of a perinuclear F-actin ring as illustrated in (a). Values are mean ± SD, n > 450/treatment. **p = 0.01, ***p = 0.001, Welch’s t-test (c,d,e and f) Abnormal accumulation of myosin-2A and active myosin (p-MLC) at the base of blebs (c, green arrowhead and f) and around nuclear extensions (d, white arrowhead) in Cof/ADF siRNA treated HeLa cells (c,d and f) and RPE-1 cells (e). Images are of confocal z-projections. Boxed region (e), shown in insets, depicts a single apical z-plane. (g) Quantification of relative pMLC fluorescence intensity to non-bleb or plasma membrane bleb cortical sites. Values are mean ± SD, n > 30 cells/treatment. (h) Representative MLC immunoblots with lamin B as a loading control. (i) Quantification of nuclear structural defects in RPE-1 cells siRNA treated along with ectopic expression of proteins as indicated. Values are mean ± SD, ROCK-CA n = 94 and n > 270 for all other treatments. *p < 0.05, **p = 0.01, ***p = 0.001, ns, not significant, one-way ANOVA and Dunnett’s post hoc test, relative to control. (j) Immunofluorescence labeling of F-actin and lamins in ROCK-CA expressing RPE-1 cells. Boxed region is magnified in last panel. Bars, 10 µm.

Fig 3: Quantitative analysis of LMNA ChIP-seq data using EDD. (a) IGV browser views of domains enriched in LMNA discovered by EDD (EDD peaks) on chromosome 8 in AD04DF (2 replicates) and LDF. LMNA profiles are shown as log ChIP/Input ratios. Genes and FPKM values (AD04DF) are also shown. (b) LMNA peaks (LADs) identified by RSEG, BroadPeak, SICER and PeakRanger in AD04DF (both ChIP-seq replicates). DamID-derived LMNA LADs in HT1080 cells (11) are also shown (bottom track). (c) Median peak length detected by EDD, BroadPeak, SICER and RSEG in AD04DF_rep1 and rep2, and in LDF (*P < 10-75 relative to BroadPeak, SICER and RSEG; Wilcoxon rank-sum test; see Supplementary Table S3 for W- and P-values).

Fig 4: LINC complex disruption in multiple human cell types induces contrasting F-actin cytoskeletal anomalies. (A, B) Basal confocal immunofluorescence images of HeLa cells labeled for F-actin (phalloidin) and myosin-2A and quantification of F-actin phenotypes. Arrow depicts cell with decreased numbers of stress fibers (SF) and the presence of F-actin condensates. Insets show enlargements and merged images (bottom row) of boxed region depicting rings of myosin-II surrounding interconnected F-actin asters. Values are mean ± SD, control n = 1192, LMNA siRNA n = 473. (C–G) Confocal immunofluorescence images of RPE-1cells siRNA treated for depletion of LMNA (C, D) and Sun1 (E–G). Numbered boxed regions (C, bottom row) are shown enlarged (D). Cells with increased SF (E), disrupted SF network (F), relative to control (C) are illustrated. Boxed region (F) is enlarged in G. (H) Immunofluorescence images of U2OS cells. Boxed region is enlarged in last column and shows a disrupted network of actomyosin asters. (I) Quantification of total abnormal F-actin phenotypes in RPE-1 cells, as classified in B. Values are mean ± SD, control n = 180, Sun1 siRNA = 163. (J–N) Immunofluorescence images of normal human fibroblasts (J), RPE-1 (K–M), and U2OS (N). Increased SF formation (K), stress fiber loss (L, M), and disrupted stress fiber network (N) are shown. Green and blue arrows (J) depict regions of increased thick SF and stress fiber loss, respectively. Boxed region (N) is enlarged in subsequent columns. (O) Quantification of total abnormal F-actin phenotypes in RPE-1 cells, as classified in B. Values are mean ± SD, control n = 377, nesprin1 (Nesp1) + nesprin2 (Nesp2) siRNA = 256. Inc, increased; Dis, disrupted; Dec, decreased; F-act, F-actin; M2A, myosin-2A. * p < 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ns, not significant, Welch’s t test. Bars, 10 μm except where noted.

Fig 5: Comparative 3D SIM imaging of the localization of AKTIP in non-transformed laminopathic cells (A) Projections of an extended section of non-transformed wild type, HGPS and EDMD2 patient derived fibroblast nuclei labelled with anti-lamin A/C and anti-AKTIP antibodies, viewed from orthogonal planes and magnified sections. B Pearson correlation coefficient to measure the co-localization between AKTIP and lamin A/C in wild type, HGPS and EDMD2 cells. C Projections of an extended section of non-transformed wild type, HGPS and EDMD2 nuclei labelled with anti-lamin B1 and anti-AKTIP antibody, viewed from orthogonal planes and magnified sections. D Percentage of AKTIP positive rim in wild type, HGPS and EDMD2 cells. In B and D each dot corresponds to a nucleus. A minimum of 7 nuclei were counted for each cell type. Mean ± SEM are shown. ***p < 0.001 in unpaired Student t-test