a) Lysates of Jurkat T cells overexpressing ATG5 or treated with etoposide (48-h cultures) were immunoprecipitated with anti-ATG5 or antisurvivin antibodies.f8(a) Lysates of Jurkat T cells overexpressing ATG5 or treated with etoposide (48-h cultures) were immunoprecipitated with anti-ATG5 or antisurvivin antibodies. A physical interaction between ATG5 and survivin, absent in untreated or GFP-expressing control cells, was detected reciprocally. Full-length immunoblots are provided in Supplementary Fig. S12. (b) Jurkat T cells overexpressing ATG5-ΔNES exhibited no ATG5/survivin molecular interaction. (c) The ATG5-K130R mutant did show coprecipitation of survivin with ATG5. (d) Calpain-mediated, truncated ATG5 (tATG5), that is, the N-terminal part of ATG5, failed to bind survivin. The ATG5-K130R mutant was the control. (e) Confocal microscopy. MDA-MA-231 cells were etoposide treated or transduced as indicated (48-h cultures) and stained. In untreated cells, survivin and ATG5 were in the cytosol with no colocalization. ATG5-transduced cells exhibited ATG5/survivin colocalization in the nucleus. ATG5-ΔNES-transduced cells showed survivin, but not ATG5, in the nuclei of cycling cells. Numerical analysis was performed and Pearson's correlation coefficients are indicated in the images. Scale bar, 10 μm. Right: Statistical analysis (analysis of variance) is presented in which the results of ten representative cells within each group were integrated. Values are means±s.d. (f) The physical interaction between Aurora B and survivin was analysed reciprocally and reduced in cells expressing high levels of nuclear ATG5. Neither ATG5 nor HSP90 were detectable in the Aurora B-containing immune precipitate, but ATG5 molecularly interacted with survivin. Results representative of three independent experiments. (g) Confocal microscopy. HeLa cells treated with etoposide or transduced as indicated (48-h cultures) were stained with centromere-specific antiserum and anti-Aurora B (top panels) or antisurvivin antibodies (lower panels). In untreated or ATG5-ΔNES transduced cells, both Aurora B and survivin were recruited to prometaphase centromeres and located at the central spindle during anaphase where they no longer colocalized with centromeres. In contrast, etoposide-treated or ATG5-transduced cells recruited much less Aurora B to prometaphase centromeres. Such recruitment, however, was observed during anaphase. Compared with cells in normal mitosis, survivin, although reduced, was recruited to centromeres in prometaphase, but, similar to Aurora B, remained attached to centromeres during anaphase. Such mislocation of Aurora B and survivin was associated with severe chromosome alignment and segregation defects. Colocalization images were prepared using Imaris software. Numerical analysis was performed and correlation coefficients are indicated. Scale bar, 2 μm. (h) Confocal microscopy. Sections of lung (non-small-cell lung cancer) and oesophageal carcinoma stained for ATG5 and survivin expression. In untreated patients, ATG5 expression in cancer cells was slight and mainly cytosolic. Following radio- and/or chemotherapy, ATG5 was preferentially expressed in the nucleus and colocalized with survivin. Overlay images present the average Pearson's correlation coefficient as calculated with Imaris. Results are representative of three independent experiments. Scale bar, 10 μm.