Figure 3. ATG5 is necessary and sufficient for anticancer drug-induced mitotic catastrophe.

(a) Immunoblotting. Jurkat T cells were cultured as indicated for 48 h. Only under conditions of ATG5 induction was evidence of increased autophagic activity obtained. Monomeric ATG5 (shown under the dotted line) was detected after a longer exposure time compared with the ATG5 conjugated with ATG12. Results are representative of three independent experiments. (b) Morphological analysis. Jurkat T cells were cultured as in a. Values are means±s.d. of three independent experiments. (c) Morphological analysis. Jurkat T cells were treated with the indicated drugs or transduced with lentivirus ATG5, ATG5-K130R, Beclin 1 or control constructs for 48 h. Enlarged and multinucleated cells were seen as a consequence of ATG5 or ATG5-K130R lentiviral gene transfer or of etoposide treatment. Increased Beclin 1 levels or rapamycin treatment had no such effect. Representative examples of morphology are shown. Scale bar, 10 μm. Right: statistical analysis of the data. At least 100 cells were counted for each condition. Values are means±s.d. (n=3). (d) Immunoblotting. Enforced expression of ATG5, Beclin 1, or ATG5-K130R following lentiviral gene transfer. (e) Time-lapse microscopy. HeLa H2B–mCherry–α-tubulin–EGFP cells were transduced by lentiviral gene transfer as indicated. Only ATG5-overexpressing cells demonstrated evidence for mitotic catastrophe (see also Supplementary Movies). As with the GFP control, the Beclin 1 construct also included GFP, hence the intensity in green was very high. Scale bar, 10 μm.