Lysosome damage induces LC3A lipidation independent of canonical autophagy. (A) Representative time-lapse confocal z stack images of MCF10A cells expressing GFP-LC3A treated with LLOMe (250 μM) or GPN (200 μM). Scale bar, 10 μm; h:min. (B) WT and ATG13 KO MCF10A cells expressing GFP-LC3A were treated with LLOMe (250 μM, 20 min) or PP242 (1 μM, 1 h). Western blotting was performed to probe for GFP-LC3A (I and II forms are marked). (C and D) Representative confocal images of GFP-LC3A in (C) WT and (D) ATG13 KO MCF10A cells treated with LLOMe (250 μM, 20 min) or PP242 (1 μM, 1 h), after pretreatment with Vps34 IN-1 (5 μM) or BAPTA-AM (10 μM). Scale bar, 10 μm. (E) Representative confocal images of WT and ATG13 KO MCF10A cells treated as in C and D, and stained for WIPI2. Scale bar, 10 μm. (F) Quantification of WIPI2 puncta from E. >100 cells were analyzed from eight fields of view in two experiments. ****, P < 0.0001, unpaired t test. Source data are available for this figure: SourceData F1.