Nuclear abnormalities in cells from old individuals. (A) Immunofluorescence microscopy on primary dermal fibroblasts from young (7 y) and old (87 y) healthy individuals and a HGPS patient. Scale bar, 10 μm. DAPI, 4′,6′-diamidino-2-phenylindole. Intensity distributions of the average fluorescent signal for (B) Tri-Me-K9H3 and (C)HP1γ in fibroblasts from healthy individuals of indicated age and a HGPS patient. (D) Quantitation of cells showing reduced amounts of Tri-Me-K9 in passage-matched population doublings (PD) 19 cell lines from healthy individuals of indicated age and a HGPS patient (). (E and F) Reduction of Tri-Me-K9 H3 over cell passage. Best linear fits are shown. (G and H)Increased DNA damage in passage-matched (PD20) cell lines from healthy individuals of indicated age and a HGPS patient detected by antibody to H2AX. Scale bar, 20 μm. N > 200.

Sporadic use of exon 11 LMNA cryptic splice site in cells from healthy individuals. (A)Comparison between the consensus donor splice sequence, the wild-type LMNA cryptic splice site in exon 11, and the mutated cryptic splice site in HGPS patients. Forward slash indicates the splice junction. (B)Schematic representation of splicing events (dotted black lines) involving LMNA exon 11 and exon 12. The cryptic splice site (dark box) and the position of different primer sets used for RT-PCR are indicated. RT-PCR analysis of wild-type and HGPS fibroblasts using (C) primers detecting both the full-length (F) and the truncated (T) LMNA isoforms or (D) primers specific for Δ150 LMNA (Δ ex11-ex12). Control ex8-ex9 primers detect all LMNA transcripts. Samples were analyzed in duplicate. Plasmids containing either full-length or truncated lamin A cDNA were used as controls for specificity of Δ150 LMNA detection. (E) Schematic representation of LMNA splicing reporter (). The cryptic splice site (dashed box) is indicated. (F) Immunofluorescence microscopy and (G) fluorescence-activated cell sorting analysis of fibroblasts from a healthy individual transiently transfected with the indicated reporter minigene or untransfected. Scale bar, 60 μm. Quantitative RT-PCR analysis of (H) wild-type fibroblasts and (I) tissues from healthy individuals of indicated age and a HGPS patient. Δ150 LMNA RNA detected by using Δex11-ex12 primers relative to total LMNA RNA detected by using ex8-ex9 primers is shown.

Accumulation of lamin A/C at the lamina in cells from old individuals. Western blot analysis of total protein extract and detergent insoluble fraction of fibroblasts from (A)healthy individuals of indicated age, a HGPS patient, and mouse fibroblasts and (B) from liver from healthy individuals probed with antilamin A/C. Δ50 lamin A is not detected in mouse fibroblasts. A fourth protein consistent with Δ10 lamin A () is also detected. Equal loading was verified by Coomassie staining of the blot. (C and D) Immunofluorescence confocal microscopy on passage-matched (PD21) fibroblasts. (D) Co-staining of fibroblasts from an old individual. Scale bar, 10 μm. Lamin A/C signal shown in intensity pseudocolors in upper right image. Arrowheads indicate nuclei with depleted nucleoplasmic lamin A/C and reduced amounts of HP1γ and Tri-Me-K9H3. (E) Quantitation of percentage of cells showing depletion of nucleoplasmic lamin A/C (average fluorescence intensity ratio nucleoplasm/lamina < 0.2) in passage-matched (PD21) cell lines from healthy individuals of indicated age and a HGPS patient.

Nuclear defects in cells from old individuals are caused by use of the LMNA exon 11 cryptic splice site. (A) Immunofluorescence microscopy on fibroblasts from an old healthy individual after treatment with either Exo11 oligonucleotide or a control scrambled oligonucleotide. Scale bar, 60 μm. (B and C) Quantitation of phenotypic rescue upon treatment with Exo11 oligonucleotide. (D) Quantitative RT-PCR analysis of HeLa cells and cell lines from young and old donors upon treatment with either Exo11 oligonucleotide or a control scrambled oligo-nucleotide. Values represent averages ± SD from a representative experiment. Statistical significances of the differences compared with mock-treated cells from the old donor are indicated. One asterisk indicates P < 0.1. Two asterisks indicate P < 0.05. (E) Quantitation of BrdU-positive cells in cell lines from young and old donors upon treatment with either Exo11 oligonucleotide or a control scrambled oligonucleotide. Values represent averages ± SD from three independent experiments.