644 mandibula, mottled cutaneous pigmentation, partial lipo- dystrophy (type A pattern), and insulin resistance (Novelli et al. 2002). The disorder is caused by recessive mutations of the LMNA gene on chromosome 1q21.2 encoding for A-type lamins, including lamin A, lamin C, lamin A delta 10, and lamin C2 obtained by alternative RNA splicing (Maraldi et al. 2011). Lamin A forms polymers at the nuclear lamina with lamin C. While lamin C is produced as mature protein, lamin A is translated as a precursor protein, which under- goes four steps of post-translational modiﬁcations, includ- ing farnesylation, double endoprotease cleavage and carboxymethylation (Maraldi et al. 2011). These modiﬁ- cations occur at the C-terminal CaaX motif, a sequence shared by farnesylated proteins, in which C is cysteine, the target of protein farnesyl transferase which catalyses prel- amin A farnesylation. In human prelamin A, the aaX sequence consists of a serine, an isoleucine and a methionine (SIM residues) and the methionine directs the addition of the 15 Carbon farnesyl residues to cysteine. Following farnesylation, the aaX tripeptide is cleaved by ZMPSTE24 (zinc-dependent metalloproteinase Ste24 homolog) or RCE1 (Ras converting enzyme 1) and the C-terminal cysteine was carboxymethylated by the carb- oxymethyltransferase Icmt. The second ZMPSTE24-med- iated cleavage of 15 amino acids at the C-terminus of prelamin A leads to removal of the farnesyl residue and yields mature lamin A (Dominici et al. 2009). Prelamin A processing is altered in laminopathies featuring premature aging and/or lipodystrophy, including Hutchinson–Gilford progeria (HGPS), Werner syndrome, restrictive dermopa- thy, familial partial lipodystrophy (FPLD2) and MADA, as well as in mandibuloacral dysplasia associated with mutations of the ZMPSTE24 endoprotease gene (MADB) (Maraldi and Lattanzi 2007). Prelamin A was postulated to be toxic for the cells and its toxicity has been attributed to the farnesylated residue. In agreement with this hypothesis, drugs impairing protein farnesylation have been shown to ameliorate the nuclear morphological abnormalities in laminopathic cells accumulating prelamin A and the whole phenotype in Zmpste24 mice (Davies et al. 2011). It has been shown that reducing mutated prelamin A levels in progeria cells by splicing correction restores heterochro- matin markers (Scafﬁdi and Misteli 2005). Moreover, we previously showed that in progeria cells accumulating farnesylated prelamin A, chromatin organization and function can be recovered by treating with mevinolin (an inhibitor of the hydroxymethyl-glutaryl-synthase eventu- ally impairing prelamin A farnesylation) in combination with the inhibitor of histone deacetylases trichostatin A (TSA) (Columbaro et al. 2005). In the present study, we determine the post-translational modiﬁcations harbored by prelamin A in MADA cells and the effects of the treatment 123 Histochem Cell Biol (2012) 138:643–651 with mevinolin alone and in combination with TSA on heterochromatin. Here, we show that low passage ﬁbro- blasts from MADA patients accumulate farnesylated prel- amin A. However, at high passage number, full-length prelamin A, possibly in its farnesylated and non-farnesy- lated forms, is detected in cells. The examined drug treatments appear to be effective in reducing heterochro- matin defects in low passage cells only, possibly depending on the relative amount of prelamin A forms which are accumulated. Recovery of the cellular phenotype is dem- onstrated by changes in altered nuclear markers, such as trimethylated histone H3K9. Moreover, the highly disorganized lattice formed by the nuclear envelope protein SUN2 in MADA nuclei (Mattioli et al. 2011) is rescued by treatment with mevinolin, indi- cating that the altered pattern of SUN2 distribution in the nuclear envelope of MADA ﬁbroblasts is caused by farn- esylated prelamin A accumulation. Materials and methods Cell cultures MADA skin ﬁbroblasts were obtained from patients car- rying the homozygous R527H LMNA mutation that has been previously reported (Filesi et al. 2005; Lombardi et al. 2007). Control skin ﬁbroblast cultures were obtained from skin biopsies of healthy patients (mean age 24) undergoing orthopedic surgery, following a written consent. The pro- tocol had been approved by the local ethical committees. Cell cultures had been established and maintained in Dul- becco’s modiﬁed Eagle’s medium supplemented with 10 % fetal calf serum (FCS) and antibiotics. The experiments were performed at passages 8 or 14 (low passage samples) or 25 (high passage samples). Drug treatments Impairment of farnesylation and further processing of prelamin A was achieved by the use of mevinolin, an inhibitor of the hydroxymethyl-glutaryl-synthase, applied at 25 lM concentration for 18 h. When speciﬁed, follow- ing mevinolin administration, cells were treated with 2.5 lM trichostatin A (TSA), an inhibitor of histone deacetylases, for additional 24 h. Antibodies Antibodies employed for Western blot analysis or immuno- ﬂuorescence labelling were: anti-lamin A/C, goat polyclonal (Santa Cruz, SC-6215, used at 1:1,000 dilution for the Wes- tern blot analysis); anti-prelamin A, goat polyclonal (Santa