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. 2022 Aug 16;13:4804. doi: 10.1038/s41467-022-32505-8

Fig. 6. Mannose protects mitochondrial function by reducing the abnormal release of cathepsin B from lysosomes.

Fig. 6

a–e NCM460 cells were stimulated with 2.0% DSS with or without mannose for 24 h, and the protein level of cathepsin B in the cells was determined by western blotting (a) and immunofluorescence staining (Scale bar = 20 μm) (b). The activity of cathepsin B in the culture medium was also evaluated (c). The expression levels (d) and activities (e) of cathepsin B in the lysosomal fraction and other remaining cytoplasm of NCM460 cells were assessed. f–i NCM460 cells were pretreated with CA-074 (10 μM), a selective inhibitor of cathepsin B in live cells, before DSS and mannose stimulation. f The expression of tight junction proteins in the cells was determined by western blot analysis. g, h Mitochondrial morphology, mitochondrial ROS production, and mitochondrial membrane potential were detected by MitoTracker, MitoSOX, and JC-1 probes, respectively. Scale bar = 20 μm. ImageJ software was used to analyze the images. i The levels of mitochondrial PDHK1 and PDH were evaluated by immunoblotting analysis. j PDH+/+ and PDH−/− NCM460 cells were stimulated with 2.0% DSS with or without mannose for 24 h, then the expression of activated cathepsin B and tight junction proteins was determined by western blotting analysis. k Cathepsin B+/+ and Cathepsin B−/− NCM460 cells were stimulated with 2.0% DSS with or without mannose for 24 h, then the expression of tight junction proteins was determined by western blotting analysis. *p < 0.05, **p < 0.01, ***p < 0.001. Data were analyzed by an unpaired Student’s t-test (a–k) and shown as means ± SD. Data from one representative experiment of three independent experiments are presented. Source data are provided as a Source Data file.