Figure 4.
Reduced levels of farnesylated prelamin A affect SUN1 expression and nuclear positioning in myotubes. (A) Differentiating myoblasts (day 6 post-confluence) were left untreated (untreated) or treated with mevinolin for 48 h (mevinolin). Cell cultures were fixed at day 8 post-confluence. Highly differentiated myotubes (more than 12 nuclei) are shown in a, b, e, f, i, l, o and p. Myotubes with less than six nuclei are shown in c, d, g, h, m and n. Farnesylated prelamin A was detected using 1188-2 antibody (a, b, red). Full-length prelamin A (non-farnesylated prelamin A) was detected using 1188-1 antibody (c–f, red). SUN2 was detected using anti-SUN2 antibody (g–l, red). SUN1 was detected using anti-SUN1 antibody (m–p, red). Caveolin 3 (green) was stained as a marker of myogenic differentiation. Bar, 10 μm. Clustering of myotube nuclei (more than three agglomerated nuclei in unordered distribution on one side of the myotube, with myotube areas devoid of nuclei) is observed in mevinolin-treated cells (right lane). Misshapen nuclei (nuclei with blebs, invaginations, or enlarged nuclei) are also observed (d, f, h). (B) Statistical analysis (mean values±S.D. of counts performed in three different samples). Counting was performed in 100 myotube nuclei per each sample. Misshapen nuclei (misshapen) or nuclei belonging to a cluster (clustered) were counted in myotubes left untreated or treated with mevinolin (upper panel). Nuclei showing nuclear pole enrichment of SUN2 (detected using anti-SUN2 antibody) or prelamin A (detected using anti-prelamin A 1188-2 antibody in untreated cells and anti-prelamin A 1188-1 antibody in mevinolin-treated cells) were counted (middle panel). SUN1 or farnesylated prelamin A fluorescence intensity, labeled using anti-SUN1 or anti-prelamin A 1188-2 antibody, respectively, was determined using the NIS software and registered for 100 nuclei (lower panel). All the differences between untreated and mevinolin-treated samples were statistically significant (P<0.05). (C) Western blotting analysis of SUN1, SUN2, prelamin A, and lamin A/C in untreated or mevinolin-treated (+mev) myotube cultures. Prelamin A was detected using anti-prelamin A 1188-1 antibody directed to the full-length protein. Emerin is used as a loading control. Molecular weight markers are reported in kDa. Densitometric values are reported as percentage of values registered in untreated samples and represent means±S.D. of three different experiments