Figure 7.

Uncoupling Actin Cytoskeleton from the LINC Complex Rescues Nuclear Deformation

(A) IF staining for Lamin A/C of ADF-null SCCs treated with siNT or siCFL1 alone or in conjunction with siRNAs targeting Nesprin-2G. White arrows designate normal nuclear morphology, blue arrows moderately misshaped, and yellow arrows enlarged nuclei. Green dashed arrow indicates a gap (white bar) between the nuclear envelope (NE) and the closest actin fibers.

(B) qPCR showing knockdown efficiency of siRNAs targeting Nesprin-2G. The data were normalized against cells treated with 64 nM siNT and are presented as mean ± SD. The primers are shown in Table S1.

(C) Phase contrast images of cells treated as in (A) (note that images of cells treated with siNT or siCFL1 [left panels] are the same as in Figure 5A, as they were part of the same experiment).

(D) Quantification of cells that displayed non-fragmented nuclei. Results are presented as mean ± SEM (n = 3). Greater than 150 cells were counted per condition per repeat. ^∗∗∗one-way ANOVA with Tukey’s multiple comparison test; p value ≤ 0.0001.

(E) Quantification of the distance between the NE and the closest visible actin fibers in cells treated with siCFL1 alone or together with siNesprin-2G (see green dashed arrow in A). Thirty-six cells were quantified from greater than or equal to nine fields of each treatment (n = 2). Measurements were performed in ImageJ using images acquired with confocal microscopy; they were evenly spaced around the periphery of the nuclei and plot displays all individual measurements (distance in μm) ± SD. ^∗∗∗Mann-Whitney p value < 0.0001.

(F) Quantification of F-actin levels. Greater than or equal to 60 cells were quantified per condition (n = 3). Data are presented as mean ± SEM. ^∗∗∗Kruskal-Wallis ANOVA with Dunn’s multiple comparison test; p value ≤ 0.0001.

(G) Knockdown efficiency of siRNAs targeting SUN1 (64 nM; siSUN1) was tested by immunoblotting.

(H and I) Phase contrast (left panels) and IF (right panels) images of ADF-null SCCs treated with siNT or siCFL1 alone or together with siSUN1 (H) and quantification of cells with non-fragmented nuclei following these interventions (I). Data are presented as mean ± SEM (n = 3). Greater than 150 cells were counted per treatment per repeat. ^∗∗∗unpaired t test; p value < 0.0001.

The scale bars represent 60 μm for phase and 20 μm for IF images. Nuclei and F-actin were counterstained with DAPI and phalloidin, respectively.