(a) RNAse protection assay on control cells (CRL-1474) and HGPS cells (AG11498) subjected to 3 electroporations with oligonucleotide or mock treated. A longer exposure of the gel is shown for CCL8, MMP3, HAS III and MMP14 due to the low expression level of those genes compared to STAT3, TIMP3, L32 and GAPDH. (b) Quantitation of gene expression levels. Values represent fold differences of the indicated mRNAs levels between untreated and treated HGPS cells and control cells. Values are averages from at least 3 independent experiments ±S.D.

(a–d) Immunofluorescence microscopy on primary dermal fibroblasts from a healthy control individual (a) and a HGPS patient (AG01972) untreated (b) or treated with oligonucleotide (c). Cells were stained with DAPI (blue) and antibodies (red) against the indicated proteins. The percentage of cells showing aberrant phenotype is indicated. Scale bar: 10 μm. (d) Low magnification images of untreated and treated HGPS cells. Scale bar: 35 μm. (e) FRAP analysis of wt GFP-lamin A in HGPS cells upon splicing correction. A nucleus was imaged before and during recovery after the bleach pulse. Scale bar: 5 μm. (f) Kinetics of recovery of the fluorescence signal in the whole bleached area compared to control and untreated HGPS cells. The statistical significance of the differences between the curves is indicated.

(a) Schematic representation of the region of lamin A pre-mRNA targeted by exo11 antisense oilgonucleotide. The predicted splicing events (dotted line for the normal splicing, dashed line for the aberrant splicing) and the position of exo11 oligonucleotide (black bar) are indicated. (b) RT PCR analysis of control fibroblasts and lymphocytes and HGPS fibroblasts and lymphocytes subjected to 2 sequential electroporations with oligonucleotide or mock treated. (c) Quantitation of exo 11 (black symbols) and scrambled (with symbols) oligonucleotides titration in different cell lines. All values represent averages from 5 independent experiments ±S.D. (d) Schematic representation of the different fragments of lamin A (black bars) and lamin C (gray bar) cDNAs amplified by RT-PCR. (e) RT-PCR analysis and (f) quantification of the indicated fragments after oligonucleotide titration. The intensity of each band was normalized to the intensity of the corresponding β-actin band. (g) Western blot analysis of control cells and HGPS fibroblasts subjected to 3 sequential electroporations with oligonucleotide or mock treated, analyzed 6 days after the first delivery.

(a–c) Immunofluorescence microscopy on primary dermal fibroblasts from a healthy control individual (AG08469; population doublings: 25–30) (a) and a HGPS patient (AG01972; population doublings: 25–30), untreated (b) or transfected with wild type lamin A and GFP as a transfection marker (c). Cells were stained with DAPI (blue) and antibodies (red) against the indicated proteins. Four cells (indicated by arrowheads) with normal staining respectively for lamin B, LAP2, HP1α and Tri-Me-K9 are shown in panel b to directly compare the cellular level of the proteins in unaffected and affected cells. The percentage of cells showing aberrant phenotype is indicated. Scale bar: 10 μm. (d) FRAP analysis of wild type GFP-lamin A in living control and HGPS cells. Nuclei were imaged before and during recovery after the bleach pulse. Scale bar: 5 μm. (e) Kinetics of recovery of the fluorescence signal in the whole bleached area. The statistical significance of the difference between the two recovery curves is indicated. (f) Immunofluorescence microscopy on primary dermal fibroblasts from a healthy control individual (AG08469; population doublings: 25–30) transfected with GFP-Δ50 lamin A. Cells were stained with DAPI (blue) and antibodies (red) against lamin A/C, lamin B, LAP2 proteins. The green fluorescent signal from GFP-Δ50 lamin A is overlaid with the DAPI signal. Scale bar: 10 μm.