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. 2021 Aug 5;29(1):147–155. doi: 10.1038/s41418-021-00843-7

Fig. 3. Caspase-8 aggregates and is activated within the cytosol following proteasome inhibition.

Fig. 3

A LC3 I and II as well as p62 amounts in HCT-116 (Bax/Bak)−/− cells treated with 100 nM bortezomib (±50 µM z-VAD-fmk) for 48 h. β-actin and GAPDH served as loading controls. B GFP-LC3 distribution in HCT-116 (Bax/Bak)−/− cells treated as indicated. Nuclei were stained with Hoechst 33342. Scale bar = 10 µm. C Ultrastructural changes in HCT-116 (Bax/Bak)−/− cells treated as indicated. Condensed mitochondria (black arrows), aggresomes (black arrowheads), dilated ER (gray arrows). Aggresomes appear as optically dense but otherwise largely unstructured subcellular protein accumulations. Scale bars = 5 µm. D Aggresome staining in HCT-116 (Bax/Bak)−/− cells treated with 100 nM bortezomib. Nuclei were stained with Hoechst 33342. Scale bar = 10 µm. Data show mean values and sd from n equals three independent fields of view with >30 cells each (**p < 0.01, t-test). E Localization of caspase-8 and aggresomes in HCT-116 (Bax/Bak)−/− cells expressing LC3-GFP. Cells were treated with 100 nM bortezomib and 5 µM Q-VD-OPh for 48 h, fixed, and immune-stained for aggresomes and caspase-8. Scale bar = 10 µm. Overlay-line scan and Pearson’s correlation analysis demonstrates preferable co-localization of caspase-8 and aggresomes (bar graphs shows mean values and sd from n = 12 cells; *p < 0.05, paired t-test).