Supplemental Figures

This article contains the following supporting material:

• Supplemental Figure 1.pdf - Cells expressing indicated constructs (as in figure 4E) were analyzed by confocal microscopy. Representative images of untreated cells or treated with TNFα with or without E₂ pretreatment are shown. White arrows in all the panels show transfected (green) cells and the corresponding cellular/nuclear structures in DIC images. Note “shrinking” of nuclei typical of apoptotic cells as well as appearance of “cellular fragments” in panels 2, 5, 6 and 7 may that be related to apoptotic bodies described in the literature (Soini et al., 1998). Black arrows in panels 6 and 7 indicate untrasfected cells that maintain the normal cellular morphology in the presence of TNFα+E₂. Expression of EGFP alone in control cultures did not induce apoptotic changes (white arrows in the insert).
  References Soini,Y., Paakko,P., Lehto,V.P. (1998). Histopathological evaluation of apoptosis in cancer. Am.J.Pathol. 153, 1041-1053.
  
• Supplemental Figure 2.pdf - Cells grown on coverslips (as triplicates) were co-transfected with EGFP together with indicated constructs. Cells were maintained in complete medium for 48hrs after the transfection, fixed in 3% paraformaldehyde, stained with Hoechst 33342 and mounted on to glass slides. In RasN17 trasfected cultures the floating cells were collected and cyto-spun on to glass slides. At these time points the amount of floating cells observed in other cultures were negligible.
  Nuclear morphology of cells with green fluorescence was scored either as normal or apoptotic using fluorescence microscopy.
  

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