Figure 2. .

Increased cell death in HeLa cells after transfection with Tr-J and Tr mutations. A, Apoptosis induced in HeLa cell lines after transient transfection (scale bar = 25 μm). Cell lines transfected with wild-type (Wt) PMP22 (b) show a significantly lower number of positive cells. Data from TUNEL assays revealed the presence of more TUNEL-positive cells only after transfection with Tr-J (c) and Tr (d) mutations. Negative control cells (a) transfected with empty plasmid are also shown. B, Western-blot analysis showing similar PMP22 protein levels (arrow) for both wild-type and mutants in transiently transfected HeLa cell lines. Quantitative RT-PCR experiments revealed equal amounts of mRNA in the experimental and control samples, which is consistent with the western blots (not shown). C, Representative study of the flow-cytometric analysis of apoptosis after transfection of cells with wild-type PMP22, Tr-J, and Tr. Significant differences were observed in the percentage of cells undergoing apoptosis when transfected with Tr-J and Tr, showing a higher toxic effect of those mutations on cells compared with that of wild-type PMP22 (representative data are from one of four independent experiments with comparable results). FS = flow-sorted cells; PI = propidium iodide.