Prelamin A mediated recruitment of SUN1 to the nuclear envelope directs nuclear positioning in human muscle

SUN1 and farnesylated prelamin A are recruited to the nuclear envelope in differentiated muscle cells

Bright staining of SUN1 was observed in cycling myoblasts, but it was considerably reduced in resting myoblasts (Fig. 1A arrowheads) and it started to increase in cells committed to differentiation (caveolin 3 positive mononucleated cells, Fig. 1A). Conversely, labeling of SUN2 was not significantly changed in myoblast subpopulations at any stage (Fig.1A). Unexpectedly, SUN1 fluorescence intensity was enhanced in myotubes, while SUN2 staining increased in a lower percentage of myotube nuclei (Fig. 1A). Moreover, farnesylated prelamin A was absent from cycling myoblasts, while it was detected in committed myoblasts and myotubes, throughout the differentiation process (Fig. 1 A). In fact, 1188-2 anti-prelamin A antibody, which selectively binds farnesylated prelamin A (37) (Fig. 1 B), brightly stained the nuclear envelope and, less intensely, the nucleoplasm, in all of the multinucleated cells (Fig. 1 A). Other anti-prelamin A antibodies, which were directed to non-farnesylated epitopes in the prelamin A C-terminus (Fig. 1 B), failed to stain prelamin A in human muscle cells, at any stage (Fig. 1 C-D). Although farnesylated prelamin A could not be detected in nuclear lysates, due to the low efficiency of 1188-2 antibody in Western blot analysis, the immunochemical analysis supported the finding that SUN1 levels were decreased in resting myoblasts, while they rose again in myotubes (Fig. 1E-F, statistically significant increase of SUN1 in differentiated cell cultures, p < 0.05). Intriguingly, a 200 kDa band, likely corresponding to a SUN1 dimer, was detected in myotubes, but not in cycling or resting cells (Fig. 1E) (10). SUN2 expression was not significantly changed at any stage in nuclear lysates (Fig. 1E-F). Thus, our results, in agreement with reported data (22, 38), showed modulation of SUN1 and prelamin A levels during myogenesis.

SUN2, farnesylated prelamin A and SUN1 at the nuclear poles and in tissues

Next, we compared the localization of prelamin A and SUN proteins at the nuclear envelope of human muscle cells (Fig. 2). Colocalization of farnesylated prelamin A and SUN1 was observed in committed myoblasts and myotubes (Fig. 2A). Moreover, the mean fluorescence intensity of SUN2, prelamin A and SUN1 was measured in cycling, resting cells or differentiate myoblasts (300 nuclei per sample). The statistical analysis reported in figure 2B shows parallel upregulation of proteins in myotubes. In depth evaluation of protein localization in committed myoblasts and myotubes allowed us to show that, while the nuclear envelope protein emerin was evenly distributed in the nuclear membrane, SUN2 was concentrated at one or both the nuclear poles in about 60% of nuclei (Figure 2C-G). Farnesylated prelamin A was localized along the nuclear rim, but it was enriched at the nuclear poles in more than 60% of myotube nuclei (Fig. C-G). SUN1 was enriched at one side of the nuclear envelope, possibly corresponding to the site of attachment of the microtubule-organizing center (MTOC), in 35% of myotube nuclei (Fig. 2C-G). However, double immunofluorescence staining of FLAG-tagged farnesylated prelamin A and SUN2 showed co-localization at the nuclear poles (Fig. 2D). Both statistical analysis (Fig. 2 E) and examination of fluorescence intensity profile of the nuclear envelope (Fig. 2 G), confirmed polarization of SUN2, prelamin A and SUN1 in committed myoblasts and myotubes. The nuclear poles are the extremities of the major axis of the cell nucleus. They anchor cytoskeletal proteins implicated in nuclear movement (39, 40). Thus, data here reported hint at a key role of SUNs and farnesylated prelamin A in myonuclear positioning.

Localization of SUN proteins and prelamin A was also investigated in mature muscle. We observed SUN1, SUN2 and prelamin A labeling at the nuclear envelope of myofibers (Fig. 2H). The nuclear envelope constituents were also detected at the myotendinous junctions, identified by integrin beta 1D labeling (Fig. 2H), and at the neuromuscular junctions, labeled by alpha-bungarotoxin staining of acetylcoline receptors (Fig. 2H). Colocalization of SUN2, farnesylated prelamin A and SUN1 was observed in the same nuclei in serial muscle sections (Fig. 2H).

Overexpression of farnesylated prelamin A enhances SUN1 levels

Because it had been reported that recruitment of SUN1 to the nuclear envelope is mediated by prelamin A (41, 42), while we had shown that prelamin A levels are increased in differentiating mouse muscle (22), we investigated a mechanistic role of prelamin A in SUN1 recruitment in human myoblasts.

To evaluate prelamin A effects on SUN1 recruitment, we transfected HEK293 cells with prelamin A constructs. Wild-type prelamin A (LA-WT), which is processed to mature lamin A, LA-C661M prelamin A, which cannot be farnesylated, LA-L647R prelamin A, which is farnesylated but cannot undergo endoproteolysis (Fig. 3A), were overexpressed in nuclei. In HEK293 cells, overexpression of farnesylated prelamin A caused a striking increase of SUN1 and SUN2 staining at the nuclear envelope (Fig. 3B). It is noteworthy that basal levels of SUN1 and SUN2 fluorescence intensity were very low in HEK293 cells (Fig. 3B). Western blot analysis confirmed that overexpression of prelamin A causes upregulation of SUN1 (Fig. 3C). SUN2 levels were not affected, as determined by Western blot analysis (Fig. 3C), suggesting an increased accessibility of native SUN2 to IF antibody labeling in transfected HEK293 cells. Moreover, we found that farnesylated prelamin A, but not other prelamin A constructs, co-immunoprecipitated SUN1 in vivo (Fig. 3C). However, a double prelamin A mutant (LA-L647R/R527P), carrying the pathogenetic R527P LMNA mutation that hinders SUN1 binding (43), failed to bind prelamin A (Fig. 3C), suggesting that some other interaction in the lamin A/C domain is required to stabilize prelamin A binding. Conversely, SUN2 and emerin bound any prelamin A form with the same affinity (Fig. 3C). To evaluate possible transcriptional regulation of SUN1 by farnesylated prelamin A, we analyzed SUN1 transcripts. The real-time RT-PCR analysis showed three-fold upregulation of SUN1 expression in cells overexpressing farnesylated prelamin A, but downregulation of transcripts in the presence of non-farnesylated prelamin A (Fig. 3D). We further hypothesized that prelamin A binding might stabilize SUN1 at the nuclear envelope in transfected HEK293 cells. Treatment of untransfected cells with the proteasome inhibitor MG132 or the lysosome inhibitor chloroquine increased SUN1 levels indicating that degradation of SUN1 does occur in HEK293 cells, while prelamin A accumulation mimics inhibition of the degradation pathways (Fig. 3E). Consistent with the possibility that prelamin A anchors and stabilizes SUN1 at the nuclear envelope, we found that overexpression of the double-prelamin A mutant which is unable to bind SUN1 (LA L647R/R527P, Fig. 3C) (43), while triggering SUN1 mRNA expression (Fig. 3D), failed to increase SUN1 staining in nuclei (Fig. C and F). Thus, both increased mRNA levels and protein anchorage contribute to SUN1 recruitment to the nuclear envelope.

It has been recently shown SUN1 recruitment at the nuclear envelope of progeria fibroblasts accumulating prelamin A (43), suggesting that recruitment of SUN1 by prelamin A occurs in diverse cell types. This hypothesis was supported by data obtained in human myoblasts. While overexpression of prelamin A in cycling myoblasts did not significantly affect SUN1 or SUN2 staining in the majority of nuclei (Fig. 1, supplemental material), overexpression of prelamin A mutants in resting myoblasts influenced SUN1 staining at the nuclear envelope (Fig. 3G). SUN2 fluorescence intensity was not significantly affected by prelamin A in transfected myoblasts (Fig. 3G). SUN1 labeling was increased in cells transfected with uncleavable prelamin A (LA-L647R), while a variable effect was obtained using LA-WT and LA-C661M (Fig. 3 G). We could not completely rule out that SUN1-containing complexes, more tightly associated with insoluble structures, may include non-farnesylated prelamin A, since, co-localization of non-farnesylated prelamin A and SUN1 was observed at the nuclear rim and in nuclear aggregates (Fig. 3 G). However, the whole evaluation of the above reported data allowed us to conclude that farnesylated prelamin A is the only form of the lamin A precursor recovered in normal differentiating human myoblasts and mature muscle and to suggest that prelamin A influences SUN1 anchorage at the nuclear envelope of differentiating human muscle cells.

Mislocalization of prelamin A and SUN2 in mevinolin-treated myotubes

To start addressing the relevance of prelamin A farnesylation and SUN1 modulation in human muscle, we wondered if changes in the post-translational modification harbored by prelamin A would have affected SUN1 expression and/or myoblast differentiation. Hence, we treated differentiating human myoblasts with mevinolin, an HMG-CoA reductase inhibitor known to impair farnesylation of prelamin A, thus eliciting accumulation of the non-farnesylated lamin A precursor.

In mevinolin-treated samples, we evaluated prelamin A localization, SUN1 and SUN2 staining and myonuclear distribution.

As expected, 1188-2 antibody stained untreated myotube cultures, but failed to label mevinolin-treated cells (Fig. 4A, panels a, b). Consistently, antibodies directed to full-length prelamin A brightly stained the nuclear envelope in mevinolin-treated cells, while they did not stain untreated samples (Fig. 4A, panels c-f show antibody 1188-1 staining). Non-farnesylated prelamin A was not enriched at the nuclear poles, contrary to what observed for the farnesylated form, but it was evenly distributed along the nuclear rim in most of the examined nuclei (Fig.4A, d, f). A minor percentage of nuclei showed mislocalization of non-farnesylated prelamin A and formation of enlarged aggregates (Fig. 4A, panel f). The intensity of SUN2 fluorescence staining was not or slightly affected by mevinolin treatment (Fig. 4A, panels g-l). However, enrichment of SUN2 at the nuclear poles was completely lost in treated cells (Fig. 4A, panels h, l and statistics in B). These observations suggested that a specific localization of prelamin A and SUN2 in myotubes is maintained through an intermediate binding partner recruited by farnesylated prelamin A, while other prelamin A forms and/or mature lamin A anchor SUN2 all over the nuclear envelope.

SUN1 recruitment is reduced in mevinolin-treated myotubes

Treatment of cycling myoblasts with mevinolin did not affect SUN1 levels (Fig. 2, supplemental material). However, the increase in SUN1 staining observed in control myotubes, was not detectable in 60 to 70% of myotubes subjected to mevinolin treatment (Fig. 4A, panels n, p and statistics in C). This result demonstrated that accumulation of farnesylated prelamin A, but not its unprocessed form, causes upregulation and recruitment of SUN1 to the nuclear envelope, in human myotubes. Indeed, SUN1 protein levels were reduced in cells subjected to mevinolin treatment, as determined by Western blot analysis (Fig. 4C). Overexpression of FLAG-prelamin A in human myotubes, supported these results (Fig. 1, supplemental material). In cells overexpressing wild-type prelamin A or LA-L647R prelamin A, SUN1 levels were not substantially affected (Fig. 1, supplemental material). We interpreted these results by considering that accumulation of SUN1 in the nuclear envelope (that occurs in untransfected myotubes) cannot be further enhanced by overexpression of farnesylated prelamin A. Conversely, in agreement with the effect observed in mevinolin-treated cells, overexpression of LA-C661M, which elicits accumulation of non-farnesylated prelamin A, lowered SUN1 levels in 30% of myotube nuclei (Fig. 1, supplemental material). Since accumulation of non-farnesylated prelamin A in cycling myoblasts did not influence SUN1 levels, while endogenous prelamin A was not detected at that stage, we suggest that SUN1 expression in cycling muscle cells may be regulated by other factors. Moreover, SUN2 protein levels, which were not affected by mevinolin treatment (Fig. 4C), appear to be independent of prelamin A.

Clustering of nuclei in myotubes accumulating non-farnesylated prelamin A

Myotubes treated with mevinolin showed mislocalization of nuclei into enlarged clusters at the extremities of multinucleated cells (Fig. 4A and statistics in B, p<0.05 in treated versus untreated myotubes). Further, we found unordered arrangement of myonuclei within each cluster, along with high variability of myonuclear size and morphology (Fig. 4A and statistics in B). These results indicated that farnesylated prelamin A is involved in myonuclear anchorage or incorporation within myotubes. Clustering of nuclei mostly occurred in myotubes showing well-organized sarcomeres and more than 12 nuclei (Fig. 4A, panels b, f, l, p), suggesting that farnesylated prelamin A could play a major role in the formation of secondary myotubes. Along this line, recent reports show that SUN1 and SUN2 play a critical role in nuclear positioning in mouse muscle (7). Moreover, altered organization of nuclei at the neuromuscular junctions, associated with SUN2 mislocalization, has been demonstrated in muscle from laminopathic mouse models (13). Regarding the role of prelamin A-and SUNs in nuclear positioning, we propose the following mechanism. Farnesylated prelamin A accumulation triggers upregulation of SUN1 and favors localization of SUN2 at the nuclear poles. Both SUN1 and SUN2, known to interact with centrosomal constituents (11, 44, 45), drive nuclear movement through cytoskeleton binding. In this context, a role of the prelamin A and SUN1 binding partner emerin, implicated in centrosome positioning (46) and undergoing modulation and cytoskeleton binding during myogenesis (47, 48), appears likely. However, an interplay between SUNs, prelamin A and centrosomal proteins in human myoblasts deserves further investigation.

Physiological relevance of SUN1-prelamin A interplay

Persistence of SUN1 and prelamin A in mature muscle, hinted at the existence of a molecular complex involved not only in myogenesis, but also in muscle homeostasis. To evaluate the physiological relevance of SUN1-prelamin A interplay, we examined protein distribution in human myofibers at different ages. Prelamin A and SUN1 underwent parallel modulation, with myofibers from younger individuals (4 months) showing faint or absent labeling of both proteins at the nuclear rim (Fig. 5A). SUN1 and prelamin A staining increased in adult and aged muscle. Bright staining of both nuclear constituents was observed starting at age 20 up to age 82 in all muscle and connective tissue nuclei (Fig. 5A). The modulation of prelamin A and SUN1 levels here shown during muscle development and ageing (Fig. 5A-B) is consistent with the possibility that both proteins might regulate nucleo-cytoskeleton relationships in adult to aged muscle. Importantly, it has been shown that SUN proteins are dispensable for muscle development in the embryo, while they play a key role in muscle homeostasis (7). Moreover, it has been reported that myonuclear domains are differently organized in young, adult or aged muscle (1), suggesting age-related modulation of proteins, such as SUN1, regulating nuclear positioning and cytoskeleton interplay.

Low farnesylated prelamin A and SUN1 levels in EDMD myoblasts associated with clustering of myonuclei

LMNA mutations causing muscle laminopathies may interfere with prelamin A-SUN1 interaction (43). Moreover, altered myonuclear positioning and chromatin defects have been reported in laminopathic muscle (13, 49, 50). Based on these published data and on the new findings here obtained in normal human muscle, we wondered if LMNA pathogenetic mutations might affect SUN1 expression or recruitment to the nuclear envelope. In autosomal EDMD (EDMD2) myoblasts, we observed indeed lower levels of farnesylated prelamin A and SUN1 (Fig. 5C, D), while non-farnesylated prelamin A was not detected. About 40% of myotube nuclei were negative for prelamin A and SUN1 labeling (p<0.05 in 100 counted nuclei from 3 different EDMD cultures versus controls). Importantly, highly differentiated myotubes showed nuclear clustering and misshapening (Fig. 5C, D), reminiscent of what observed in wild-type myotubes in the absence of farnesylated prelamin A (Fig. 4). In mature muscle, both SUN1 and prelamin A levels were slightly affected by LMNA mutations (Fig. 5E). Nuclear clustering, however, was observed in longitudinal fibers (Fig. 5E, F). We suggest that reduced prelamin A and SUN1 recruitment in EDMD2 primarily affects satellite cells activity and myotube formation. However, downstream effects including clustering of myonuclei may progressively accumulate in mature muscle.

Conclusion

The functional connection between the cytoskeleton and the nucleus involves proteins of the so-called LINC complex (after linker of cytoskeleton and nucleoskeleton), including SUNs and nesprins (51). It is possible that SUN proteins not only regulate nuclear anchorage, but also co-ordinate positioning of chromatin domains with respect to that of the nucleus (52, 53). This intriguing function appears to be particularly relevant in differentiating myoblast nuclei, which undergo dramatic changes in their structure, location and higher order chromatin arrangement (1). Here we show that SUN1 expression is modulated during differentiation of human muscle progenitors in a farnesylated prelamin A dependent way. Our data further hint at the existence of a molecular complex including SUN1 and prelamin A, which could be involved in adult muscle homeostasis. The interplay between prelamin A and SUN proteins is implicated in myonuclear positioning in healthy human muscle. Hence, myonuclear positioning is affected by the low levels of farnesylated prelamin A and SUN1 found in EDMD2 myoblasts. Thus, we suggest that prelamin A levels and post-translational modifications are crucial to protein functionality in myogenic progenitors.

The perspective of our study will be to identify modulators of prelamin A levels as well as other binding partners of farnesylated prelamin A and/or SUNs, relevant to the described effects in human muscle. For instance, FHL1, nesprin 1 and 2 represent good candidates, because they are involved in myonuclear positioning (44, 54) and myotube hypertrophy (3), and are indeed mutated in EDMD (55, 56).

Our results suggest that muscle atrophy or misfunctioning found in progeria (28) and mandibuloacral dysplasia (29) could be related to impaired modulation of prelamin A processing and downstream effects on SUN1 recruitment.

On the other hand, it has been recently reported that accumulation of non-farnesylated prelamin A causes cardiomyopathy in mice (57). This finding supports our observations in skeletal muscle and suggests that prelamin A-SUN1 interplay deserves investigation in cardiac myocytes. Finally, as hypothesised by diverse authors, it appears likely that mutations in SUN1 or SUN2 might be linked to laminopathies.

Cell culture and transfection

Human myoblast cultures were obtained from muscle biopsies by mechanical and enzymatic methods, as previously reported (58). Biopsies were obtained from 10 consenting patients undergoing orthopedic surgery for traumas. Biopsies from 3 EDMD2 patients undergoing diagnosis were used to establish primary myoblast cultures or cryosectioned to analyze protein localization. All the local and EU ethical issues were respected. Mutations found in EDMD2 muscle were: LMNA R190Q/R249Q, LMNA Y259D, LMNA L140P (58).

To obtain myotubes, confluent cell cultures were allowed to differentiate in culture medium (D-MEM plus 20% FCS plus antibiotics) for 8 days. Cells cultured for 3-4 days post-confluence were defined as resting cells if 4-8 myotubes per dish (P100 dishes) were counted. PCNA staining of these cultures was negative in 70% of nuclei. HEK293 cells were transfected with FLAG-tagged plasmids containing wild-type prelamin A, LA-WT, which undergoes normal maturation; LA-C661M, which cannot be farnesylated and LA-L647R, which is farnesylated but cannot undergo endoproteolysis (35) (Fig. 3A). The LA-R527P and LA-L647R/R527P prelamin A constructs (Fig. 3A), which carry the pathogenetic mutation R527P causing EDMD2 (43, 49) were also transfected in HEK293 cells. Transfections were performed using calcium phosphate buffer, whereas human myoblasts and myotubes were transfected using an AMAXA electroporator according to the manufacturer’s instructions. After transfection, cells were incubated for 24-48 hours (22).

When indicated, the proteasome inhibitor MG132 (1 μM for 24 hours) or the lysosomal activity inhibitor chloroquine-diphosphate (25 μM for 18 hours) were added to transfected HEK293 cells to study SUN1 degradation.

Accumulation of non-farnesylated prelamin A was obtained using 10 μM mevinolin (Sigma) in growth medium for 48 hours (59).

Real-time PCR

Total RNA was extracted using the TRIzol extraction kit (Invitrogen). Real-time PCR was performed on the panel of HEK293 cells transfected with prelamin A constructs (LA-WT, LA-C661M, LA-L647R, LA-L647R/R527P or the empty vector) for 24 hours. In particular, 500 ng of total RNA was reverse-transcribed with high capacity cDNA archive kit (Applied Biosystems, Foster City, CA). Gene-specific primers were designed using Beacon Designer software : SUN1 (accession # NM_001130965.2), forward 5′-CAG AAG CAC AAA CAA ATC-3′ and reverse 5′-CAC CAT CAT CAT CAA GAC-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as endogenous control (forward 5′-GAA GGT GAA GGT CGG AGT C-3′ and reverse 5′-GAA GAT GGT GAT GGG ATT TC-3′. Amplification was performed at the following cycling conditions: 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 55°C for 1 min. A SYBR Green PCR Master Mix (Applied Biosystems) was used with 5 ng of cDNA and with 300 nM primers. No template controls were run with every assay performed on ABI PRISM 7900 Sequence Detection System (Applied Biosystems). Data were normalized to GAPDH and expressed as relative amount of SUN1 mRNA in transfected cell lines compared to the empty vector transfected calibrator. All data are expressed as 2^−ΔΔCT. Data were obtained from triplicate experiments.

Immunofluorescence and immunohistochemistry

Cells were grown on glass coverslips and immunofluorescence staining was performed in samples fixed in 100% methanol for 7 minutes. Unfixed cryosections were saturated 45 minutes with 4% BSA prior to immunofluorescence reactions (58). Three different antibodies were used to detect prelamin A: antibody 1188-1 (Diatheva), which is directed against the last 15 aminoacids of the full length protein; antibody 1188-2 (Diatheva), which selectively binds farnesylated prelamin A devoid of the last three aminoacids (Fig. 3, supplemental material) (37); Sc-6214 anti-prelamin A antibody (Santa Cruz), which is directed against the 20 C-terminal aminoacids of the full length lamin A precursor (60) (Fig. 1B) All prelamin A antibodies were used at 1:100 dilution, except 1188-2 antibody which was used at 1:10 dilution. Rabbit polyclonal anti-SUN2 and SUN1 antibodies (9, 10) were from Atlas and were used at 1:800 and 1:300 dilution, respectively. Goat polyclonal anti-SUN1 was from Santa Cruz. Anti-caveolin 3 antibody (BD-Transduction) was diluted 1:30, anti-desmin (Novocastra) was diluted 1:100, anti-dystrophin (Novocastra) was incubated at 1:10 dilution. Texas red-conjugated alpha-bungarotoxin (Molecular probes) was used to label neuromuscular junctions. Anti-β1D integrin (Chemicon) was used to recognize myotendinous junctions.

Immunofluorescence microscopy was performed using a Nikon E600 epifluorescence microscope and a Nikon oil-immersion objective [100× magnification, 1.3 NA (numerical aperture)]. Photographs were taken using a Nikon digital camera (DXm) and NIS-Element BR2.20 software (Nikon). All images were taken at similar exposures within an experiment for each antibody. Image quantification was performed using NIS-Element BR2.20 software.

Images were processed using Adobe Photoshop 7.0 software (Adobe Systems).

Nuclear fractioning

To obtain nuclear fractions, cells detached with trypsin were incubated in cold hypotonic buffer containing 10mM Tris-HCl and protease inhibitors. Mechanical separation was then performed with a syringe and centrifugation of nuclei was performed at 1100 rpm for 10 minutes. Nuclei were lysed in buffer containing 50mM TRIS-HCl, 1% SDS containing or not 5% 2-mercaptoethanol. Some samples were heated at 70°C to check the stability of SUN1 dimers, while other samples were obtained in the absence of 2-mercaptoethanol (10). Lysates were subjected to SDS-PAGE and Western blot analysis as described below.

Immunoprecipitation and immunoblotting

Immunoprecipitation was performed in transfected HEK293 cells overexpressing prelamin A mutants, according to a procedure reported in previous studies (36). Briefly, HEK293 cells were lysed in IP buffer containing 50 mM HEPES/HCl (pH 7.4), 100 mM NaCl, 1.5 mM MgCl2, 1% NP-40, 1 mM dithiothreitol, 1 mM PMSF and protease inhibitors. After sonication and clarification, 500 μg of each lysate were incubated with anti-FLAG antibody overnight at 4°C. Control immunoprecipitations were performed in the presence of non-specific immunoglobulins. After addition of 30 μl of Protein A/G (Santa Cruz Biotechnology), lysates were incubated with continuous mixing at 4°C for 3 h. Immunoprecipitated protein complexes were subjected to Western-blot analysis.

Western blot analysis was performed on proteins separated by a 5-20% gradient SDS-PAGE. Nitrocellulose membranes were probed using the following antibodies: anti-FLAG (Sigma, 1:1000 dilution); rabbit polyclonal anti-SUN2 and SUN1 (Atlas, 1:200 and 1:100 dilution respectively); anti-prelamin A (1188-1, Diatheva, 1:100 dilution); anti-emerin (Monosan, 1:100 dilution); anti-myogenin (Santa Cruz, 1:100 dilution). Immunoblotted bands were revealed by the Amersham ECL® detection system. The intensity of bands was measured using a GS800 Densitometer (Biorad).

Statistical analysis

Statistical analysis was performed using Excel software (Microsoft). Statistically significant differences were determined using the Student’s t test.