*2.2. DG Deficiency Provokes Altered Localization and Decreased Protein Levels of Lamin B1*

Because lamin B1, a critical NE protein, is a β-DG-interacting partner [20], we were prompted to evaluate the impact of the lack of DG on lamin B1 distribution and protein expression. Interestingly, altered immunostaining for lamin B1 and evident nuclear deformities (invaginations) were found in DG-KO1 and DG-KO2 cells (Figure 3A). Consistently, the percentage of cells with aberrant nuclear morphology was clearly higher in DG-KO cell cultures than WT cell culture (right graph). Morphometric analysis of nuclei (nuclear area and circularly index) confirmed significant differences in nuclear shape between WT and DG-KO1 and DG-KO2 cells (Figure 3B). In line with IF/confocal microscopy images, a significant decrease in lamin B1 levels was observed in DG-KO1 and DG-KO2 cells (Figure 3C).

**Figure 3.** DG-KO cells show altered localization and decreased protein levels of lamin B1. (**A**) WT, DG-KO1 and DG-KO2 cells, seeded on glass coverslips, were fixed and immunostained for lamin B1. Nuclei were stained with diamidino-2-phenylindole (DAPI) prior to confocal laser scanning microscopy analysis (CLSM), and typical images are shown. Bar, 10 μm. Right: the bar graph shows the percentage of nuclei with invaginations, with significant differences calculated in each cell culture from three independent experiments using one-way ANOVA and Dunnett´s post hoc test; data indicate the mean ± SEM (*n* = 100 nuclei per cell culture; \* *p* < 0.05 compared to WT). (**B**) Nuclear morphometric analysis was carried out on WT, DG-KO1 and DG-KO2 cells, as described in Methods from three separate experiments; significant differences were obtained using a non-parametric Kruskal–Wallis test and post hoc Dunn´s method. Data correspond to the mean ± SEM (*n* = 100 nuclei per experimental condition; \* *p* < 0.05 compared to WT). (**C**) Lysates from WT and DG-KO1 and DG-KO2 cells were analyzed by SDS-PAGE/WB using antibodies against lamin B1 and GAPDH (loading control). Representative blots from three separate experiments are shown, with significant differences obtained using one-way ANOVA and Dunnett´s post hoc test; \* *p* < 0.05 in comparison to WT. Data indicate the mean ± SEM. Right: densitometric analysis of immunoblot autoradiograms was performed to estimate lamin B1 protein expression.
