Figure 7.

XPA stalls induction of apoptosis in old progeroid cells. A) HGPS cells at P8 (young) and P20 (old) passages were transfected with scrambled sequence (siCtrl) or an XPA-specific siRNA (siXPA). Five days after transfection, the cells were harvested. Cell survival was measured by flow cytometry using TMRE, which is an indicator of cells with functional, polarized mitochondria (left). Western blot analysis demonstrated caspase-3 cleavage and confirmed XPA-knockdown efficiency (right). B) HeLa, XPA^WT and XPA^−/− cells from a patient with xeroderma pigmentosum were transfected with plasmid expressing SSIM (a mutant lamin A that cannot be farnesylated), progerin (LAΔ50, GFP-LAΔ50), or an empty parent vector (GFP). At 24 h after transfection, the cells were harvested for analysis by Western blot analysis. The top blot was probed with an antibody against lamin A/C. The bottom blot was probed with an antibody against PARP. C) XPA^WT and XPA^−/− cells were transfected with the plasmid expressing progerin (LAΔ50) or empty parent vector (GFP). Cells were harvested 24 h after transfection. These cells also were separately treated with CPT for 6 h before assay. The caspase-3 activity was measured using a fluorogenic substrate (Ac-DEVD-AFC). The relative fluorescence units were normalized to the protein concentration of the sample.