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Fig. 5. FTI treatment of HGPS and RD cells. (A) Improvement of nuclear shapes of HGPS and RD cells by FTI treatment. The patient cells were treated with FTI and prepared for immunofluorescence microscopy as described in Materials and Methods. The nuclei were stained with DAPI. Misshapen nuclei are indicated by arrows. Photomicrographs were taken at 63x magnification. Bar, 50 µm. (B) FTI treatment blocked farnesylation of prelamin A, but did not change the production of -H2AX, a molecular marker for DSBs. In the left panel, BJ and HGPS cells were lysed in 2x SDS gel loading buffer and probed with the antibody directly against prelamin A. In the right panel, HGPS and RD cells were lysed and probed with the antibody for -H2AX. ß-actin was loaded to ensure similar amounts of samples were used for each pair of experimental groups. (C) Comet assays were performed with HGPS cells in the presence and absence of FTI as described in Materials and Methods. DSBs of 50 randomly chosen cells were quantitated as the percentage of DNA in tail. (D) Phosphorylation of Chk1 and Chk2 in HGPS cells with or without FTI treatment. The FTI treatment of BJ and HGPS and western blotting were performed as described in Materials and Methods. The phosphorylation statuses of Chk1 at Ser-345 and Chk2 at Thr-68 were determined with corresponding antibodies. ß-actin was probed as a loading control. (E) Accumulation of -H2AX in cells expressing prelamin A mutants. HeLa cells were transfected with plasmid pEGFP (empty parent vector), pEGFP-LA50 or pEGFP-LA50-SSIM, followed by western blot analysis. GAPDH was probed as a loading control.

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