(a) HeLa cells were transiently transfected with 24K and 33K Atg5 for 24 h and subsequently analysed by Hoechst staining. Values are means ± s.e.m. of three independent experiments. Bax transfection was used as a positive control. (b) Representative images after Hoechst staining demonstrating nuclear fragmentation of HeLa cells that were transfected with 24K Atg5 using lentiviral gene transfer for 24 h. Quantitative analysis of these experiments is shown in a. Control and transfected cells were loaded with Mitotracker orange and subsequently processed for cytochrome c immunostaining. Enforced expression of 24K Atg5 resulted in cytochrome c release into the cytosol and apoptosis (nuclear condensation, cell shrinkage; approximately 80% of the cells). 