Figure 5.
SaliP induces non-canonical autophagy and V-ATPase V0–V1 engagement. (a) Cartoon showing the differential effects of BafA1 and SaliP on V0–V1 association. (b) Confocal images of LTR staining in WT MCF10A cells treated with BafA1 (100 nM) or SaliP (2.5 μM). Scale bar, 15 μm. Quantification of LTR intensity. Data represent mean ± SD from eight fields of view. ****, P < 0.0001. (c) WT and ATG13−/− MCF10A cells were treated with BafA1 (100 nM) or SaliP (2.5 μM) for 1 h. Western blotting was performed to probe for LC3 (I and II forms marked) and GAPDH. Quantification of LC3II/I levels. Data represent mean ± SEM from three independent experiments. ***, P < 0.0001; *, P < 0.02, unpaired t test. (d) Representative confocal images of ATG13−/− MCF10A cells stained for LAMP1 and GFP-hLC3A following treatment with SaliP (2.5 μM) for 1 h. (e) Confocal images of entotic corpse vacuoles in WT MCF10A cells treated with monensin (100 μM) for 1 h and stained for LAMP1 and GFP-hLC3A. Scale bar, 10 μm. (f) Quantification of GFP-hLC3A recruitment to LAMP1 positive entotic corpse vacuoles (ECVs) following treatment with monensin (Mon, 100 μM), SaliP (2.5 μM), or BafA1 (100 nM) for 1 h. Data represent mean ± SEM from three independent experiments. ****, P < 0.0001; **, P < 0.008, unpaired t test. (g) Quantification of GFP-hLC3A recruitment to entotic vacuoles during inner cell death in WT MCF10A cells treated ± BafA1 (100 nM) or SaliP (2.5 μM). Data represent mean ± SEM from three independent experiments. ****, P < 0.0001, unpaired t test. (h) Quantification of duration of GFP-hLC3A recruitment to entotic vacuoles during inner cell death in cells treated ± SaliP (2.5 μM). Data represent mean ± SD from five examples. (i) Widefield GFP-hLC3 and DIC time-lapse images of entotic cell-in-cell structures in MCF10A cells treated ± SaliP (2.5 μM). Asterisks denote inner cells and arrows denote GFP-hLC3A recruitment to entotic vacuoles. Scale bar, 10 μm; time, h:min. Source data are available for this figure: SourceData F5.

SaliP induces non-canonical autophagy and V-ATPase V0–V1 engagement. (a) Cartoon showing the differential effects of BafA1 and SaliP on V0–V1 association. (b) Confocal images of LTR staining in WT MCF10A cells treated with BafA1 (100 nM) or SaliP (2.5 μM). Scale bar, 15 μm. Quantification of LTR intensity. Data represent mean ± SD from eight fields of view. ****, P < 0.0001. (c) WT and ATG13−/− MCF10A cells were treated with BafA1 (100 nM) or SaliP (2.5 μM) for 1 h. Western blotting was performed to probe for LC3 (I and II forms marked) and GAPDH. Quantification of LC3II/I levels. Data represent mean ± SEM from three independent experiments. ***, P < 0.0001; *, P < 0.02, unpaired t test. (d) Representative confocal images of ATG13−/− MCF10A cells stained for LAMP1 and GFP-hLC3A following treatment with SaliP (2.5 μM) for 1 h. (e) Confocal images of entotic corpse vacuoles in WT MCF10A cells treated with monensin (100 μM) for 1 h and stained for LAMP1 and GFP-hLC3A. Scale bar, 10 μm. (f) Quantification of GFP-hLC3A recruitment to LAMP1 positive entotic corpse vacuoles (ECVs) following treatment with monensin (Mon, 100 μM), SaliP (2.5 μM), or BafA1 (100 nM) for 1 h. Data represent mean ± SEM from three independent experiments. ****, P < 0.0001; **, P < 0.008, unpaired t test. (g) Quantification of GFP-hLC3A recruitment to entotic vacuoles during inner cell death in WT MCF10A cells treated ± BafA1 (100 nM) or SaliP (2.5 μM). Data represent mean ± SEM from three independent experiments. ****, P < 0.0001, unpaired t test. (h) Quantification of duration of GFP-hLC3A recruitment to entotic vacuoles during inner cell death in cells treated ± SaliP (2.5 μM). Data represent mean ± SD from five examples. (i) Widefield GFP-hLC3 and DIC time-lapse images of entotic cell-in-cell structures in MCF10A cells treated ± SaliP (2.5 μM). Asterisks denote inner cells and arrows denote GFP-hLC3A recruitment to entotic vacuoles. Scale bar, 10 μm; time, h:min. Source data are available for this figure: SourceData F5.

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