Figure 8.

Rescue of the Stress Response Phenotype by Transient Transfection of Nup-Coding Constructs into Affected Fibroblasts

Control and affected individual A1-derived fibroblasts were transfected by electroporation with constructs encoding the full-length wild-type hNUP214-GFP, hNUP88-GFP, a combination of both constructs or an empty GFP-expressing vector as a control. HS exposure was performed 48 h after transfection, for 2 h 43°C, followed by a 6 h recovery period. Cells were then fixed and immunostained for cleaved caspase-3 as in Figure 7. Representative images from one experiment are shown for the double NUP214/NUP88 transfection and empty vector control in control and affected individual-derived fibroblasts. DNA was stained with Hoechst 33258. Scale bar: 50 μm. A quantitative summary of cleaved caspase-3 staining intensity in GFP-expressing cells measured in three independent experiments is shown at the bottom. Cell borders were delineated from DIC images by the ZEN software and anti-cleaved caspase-3 staining was measured per cell and normalized to control fibroblasts transfected with the empty GFP-expressing vector. Each bar represents at least 90 cells from 3 independent experiments, showing the mean intensity ± SEM.