Histochem Cell Biol (2012) 138:643–651 Cruz, SC-6214, lot. J3105 used at 1:100 dilution for the immunoﬂuorescence analysis and at 1:700 dilution for the Western blot analysis); anti-prelamin A, rabbit polyclonal, raised against the last 18 aminoacids of the prelamin A sequence including the SIM sequence (antibody 1188-1, Diatheva); anti-prelamin A, rabbit polyclonal, raised against the last 15 aminoacids of the prelamin A sequence including the farnesylated cysteine residue but not the SIM sequence (antibody 1188-2, Diatheva); anti-trimethyl-H3K9, rabbit polyclonal (Upstate); anti-emerin, mouse monoclonal (Novocastra); anti-SUN1 or SUN2 rabbit polyclonal (Atlas); anti-actin, goat polyclonal (Santa Cruz). Western blot analysis Western blot analysis was done as follows: cells were lysed in Ripa buffer containing 1 % Nonidet P-40, 0.25 % sodium deoxycholate, 0.1 % SDS, 10 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMFS, 1 lM aprotinin, leupeptin, and pepstatin. Cell lysates were dilu- ted in Laemmli buffer, subjected to SDS-PAGE (6–20 %) and transferred to nitrocellulose membrane. Membranes were saturated with 4 % BSA and incubated with primary antibodies for 1 h at room temperature at 1:100 dilution. Secondary antibodies were used at 1:10,000 dilution for 30 min. Immunoblotted bands were revealed by Amersham ECL detection system. Immunoﬂuorescence Human ﬁbroblasts grown on coverslips were ﬁxed in 4 % paraformaldehyde at 4 C for 10 min and permeabilized with 0.15 % Triton X-100 for 5 min at room temperature. Alter- natively, cells were ﬁxed in absolute methanol at -20 C. Samples were incubated with PBS containing 4 % BSA to saturate non-speciﬁc binding, incubated with primary anti- bodies overnight at 4 C, and with secondary antibodies for 1 h at room temperature. Anti-prelamin A Sc-6214, anti- prelamin A 1188-1, anti-lamin A/C, anti-SUN1, anti-SUN2, and anti-trimethyl H3K9 were used at 1:100 dilution. Anti- prelamin A 1188-2 was applied at 1:10 dilution. Slides were mounted with DABCO (Sigma-Aldrich) anti-fading reagent in glycerol and observed with a Nikon E 600 ﬂuorescence microscope equipped with a digital camera. Results Carboxymethylated prelamin A is accumulated in low passage MADA ﬁbroblasts To discriminate the prelamin A form accumulated in MADA cells, we used two antibodies capable of detecting 645 different prelamin A modiﬁcations (Fig. 1). Antibody 1188-1 binds full-length prelamin A either in its unpro- cessed non-farnesylated form, or harboring the farnesyl residue (ﬁrst processing step) (Dominici et al. 2009). Antibody 1188-2 selectively detects farnesylated prelamin A devoid of the C-terminal SIM sequence (Dominici et al. 2009; Mattioli et al. 2011). Interestingly, immunoﬂuores- cence analysis performed on low passage cells revealed that antibody 1188-1 failed to label MADA cells, while antibody 1188-2 stained both the nuclear rim and inner nuclear invaginations (Fig. 1a). Antibody 1188-2 also labelled the nucleoplasm. As expected, control skin ﬁbro- blasts were negative for prelamin A staining using either antibody. At this stage, the Sc-6214 antibody, binding to any form of prelamin A, barely detected the precursor at the nuclear rim of MADA ﬁbroblasts, and it faintly stained control nuclei. Thus, we suggest that farnesylated and partially processed (ﬁrst step) prelamin A is accumulated in MADA cells at the ﬁrst cell culture passages, while other prelamin A forms are not detectable at this stage. Full-length prelamin A is accumulated in high passage MADA ﬁbroblasts Immunoﬂuorescence analysis of MADA ﬁbroblasts at passage 25 showed a markedly reduced labelling by 1188-2 antibody (Fig. 1b). The nuclear envelope and the nucleo- plasm were faintly labelled by this antibody and no staining at nuclear invaginations could be observed. On the con- trary, distinct staining with 1188-1 antibody, which binds full-length prelamin A (Fig. 2c), was observed both at the nuclear rim and at intranuclear invaginations. These prel- amin A aggregates likely represent nuclear lamina invag- inations, as suggested by previous studies (Filesi et al. 2005; Lattanzi et al. 2007; Mattioli et al. 2008). Western blot analysis of both low and high passage cells revealed that a prelamin A band was clearly detectable by 1188-1 or Sc-6214 antibody in MADA ﬁbroblast at passage 25 (Fig. 2d). These data suggest that full-length prelamin A is accumulated in high passage MADA ﬁbroblasts, while carboxymethylated prelamin A level appears to be reduced at this stage. Drug treatments rescue tri-methyl H3K9 pattern in low passage cells Cells from progeroid laminopathies are known to present chromatin defects, consisting of peripheral heterochro- matin loss and disorganization of heterochromatin sites labelled by tri-methylated histone 3 on lysine 9 (tri-H3K9) and HP1 (Columbaro et al. 2005; Filesi et al. 2005; Larson et al. 2012). Certain drug treatments including farnesyltransferase inhibitor administration or addition of 123