Figure 9

CB induced cell death. A. LCLC-103H cells were transiently transfected with CB(SC)-EYFP and the temporal expression course was continuously (Δt = 2 min) monitored beginning with early signs of fluorescence ~12 h post transfection. The process always followed the same scheme: Initially the yet weak fluorescence is distributed equally in the cytoplasm and in the nucleus. Then, fluorescent granules are formed (partly in the pseudopodia) and transported towards the nucleus passing the Golgi region. Granulation goes ahead with rapidly increasing fluorescence intensity in the nucleus. Finally, the Golgi apparatus disappears and the cell collapses within a few minutes while bubbling. Selected images taken by indicated points of a time-lapse experiment document these events. B. Mortality rates of LCLC-103 H cells transiently expressing CB(SC)-EGFP and control constructs (EGFP, CB(FLM)-EGFP) were determined by propidium iodide staining followed by FACS analysis. To meet falsifications of results that are caused by variable transfection rates (22–63%) the absolute mortality values were normalized to the respective transfection efficiency. Dead cells in consequence of the toxicity of the transfection agent and false positives attributable to autofluorescence were taken into account by subtraction of the respective control values: The number of dead cells (propidium iodide channel) amounted to ~13% in case of mock-transfected control cells; 2% of total were false positive "transfectants" (GFP channel). In comparison to CB(FLM)-EGFP, the toxicity of the CB(SC)-construct was almost twice as high (~78%).