Fig. 4

Cisd overexpression causes autophagosome accumulation and prevents autophagy. A Confocal micrographs of WT control versus Cisd overexpressing adult flight muscle via Mef2-GAL4, immunostained for p62 alongside imaging mCherry-Atg8a autophagosome reporter. B Electron micrographs of flight muscle as in A, showing multiple autophagic vesicles (inset) in proximity to disrupted mitochondria (arrowheads). C Larval neurons of WT control versus Cisd overexpressing or Atg5 knockdown (via nSyb-GAL4 driver) animals immunostained for p62 alongside ATP5a (mitochondria) and DAPI. D Immunoblot analysis of protein lysates from whole flies overexpressing Cisd (via da-GAL4) or WT controls. Blots were probed with antibodies against p62, Atg8a (LC3), Cisd (CISD2, Proteintech, 13318-1-AP) and Tubulin. Quantification of replicate blots is shown in Fig. S2A, B. E Quantification of the number of autolysosomes shown in F. Data points indicate individual animals analysed. Statistical analysis: unpaired t-test; ***P < 0.001. F Confocal microscopy analysis of adult flight muscle WT control versus Cisd overexpressing animals co-expressing the autophagy flux reporter GFP-mCherry-Atg8a driven by Mef2-GAL4. G, H Locomotor climbing assay of 2-day-old adult flies expressing the indicated transgenes (via da-GAL4). I Immunoblot analysis of equivalent samples analysed in G and H, probed for autophagy markers (p62 and Atg8a), Cisd and Tubulin. Quantification of replicate blots is shown in Fig. S2C. Statistical analyses: Kruskal-Wallis non-parametric test with Dunn’s post-hoc correction. ***P < 0.001; ****P < 0.0001. Scale bars = 10 μm for light microscopy, or indicated on image for EM