Histochem Cell Biol (2012) 138:643–651 647 Fig. 2 Treatment of MADA cell lines with mevinolin affects tri- methylated H3K9 labelling pattern. a Control or MADA3 cells at passage 8 or passage 25 were cultured in the absence or presence of 20 lM mevinolin for 18 h. Tri-methylated H3K9 (tri-H3K9) labelling pattern is shown. Clusters of tri-H3K9 are indicated by arrowheads. Bar 10 lm. NT non-treated, MEV ﬁbroblasts treated with mevinolin. b, c Statistical analysis. The graphs show the percentage of nuclei showing clustered trimethylated-H3K9 in control and MADA cells. MADA cells were left untreated or treated (black and colored bars, respectively) with increasing concentrations of mevinolin (25 lL) (Fig. 3a, b). SUN1 and SUN2 disorganization persisted in high passage MADA ﬁbroblasts (not shown). In line with the observation of a rescue in chromatin organisation by drug administration, we applied mevinolin and mevinolin/trichostatin A treatment to MADA ﬁbroblast and obtained an improvement of the labelling pattern of SUN2 in MADA nuclei that lost the honeycomb pattern in favor of a more homogenous distribution despite a persis- tent co-localization with prelamin A aggregates (Fig. 3b, c). Differently, SUN1 localization in intranuclear aggre- gates resulted unaffected (Fig. 3a). Comparable results in SUN2 pattern recovery were obtained in MADA cells independent from the number of passages in culture, suggesting that, differently from what observed for the tri-methylated H3K9, drug treatment was efﬁcient for phenotype rescue also at high passage number. However, complete rescue was not achievable as indicated by the persistence of co-localization with prelamin A aggregates for both SUN2 and SUN1 in control, mevinolin and mevinolin plus trichostatin A treatment conditions. Taken altogether, these data suggest that SUN2 is mainly affected by accumulation of farnesylated prelamin A and either alone (b) or followed by TSA (2.5 lM) (c). The study includes one control and three MADA cell lines: MADA1 (homozygous R527H LMNA mutation, age 18 years), CH-MADA (heterozygote compound R527H/V440M LMNA mutation, age 27 years), MADA3 (homozygous R527H LMNA mutation, age 50 years). Values are means of two independent experiments ± standard deviation; for each experiment a total of 100 nuclei were taken into consideration; p values indicate statistical signiﬁcance of the difference between treated and non-treated samples its reduction by drug treatment mediates partial recovery of phenotype at any stage of disease progression. Discussion The reported study shows that MADA ﬁbroblasts accu- mulate different forms of prelamin A depending on the passage number. Farnesylated prelamin A is accumulated in low passage cells, while full-length prelamin A, either farnesylated or non-farnesylated, is detectable in high passage ﬁbroblasts. We further show that drugs interfering with prelamin A processing can rescue the proper distri- bution of the heterochromatin marker tri-H3K9 and the organization of the nuclear envelope protein SUN2 in MADA ﬁbroblasts. Data so far reported in laminopathic cells strongly suggest that trimethylation of lysine 9 on histone 3 is a hallmark of chromatin defects related to lamin A muta- tions. In fact, tri-H3K9 aberrant organisation, manifested by loss of clustered distribution and reduced labelling intensity, has been reported in HGPS, RD and MADA cells 123