Inhibition of cathepsins B and L results in incomplete processing of cathepsins in lysosome, not in ER.

(A) Immunoblot analysis with E64d (E, 20 μg/mL), cathepsin B inhibitor (CBi, 20 μM), and cathepsin L inhibitor (CLi, 20 μM)-treated INS-1 cells in 30 mM glucose for 24 or 48 hr. (B) Immunoblot analysis of pancreatic islets of SD rats treated daily with CBi and CLi in 30 mM glucose for 48 hr. (C) Cytoplasmic and nuclear fractions were prepared from INS-1 cells cultured in 30 mM glucose medium with cathepsin B and L inhibitors for 48 hr then immunoblotted. (D) Localization of pro-cathepsins and cathepsins after treatment with cathepsin B and L inhibitors in 30 mM glucose for 48 hr by cytosol and mito- / lyso- (mitochondria / lysosomal) fractionation. (E) Lysosome-enrichment extraction was prepared and separated by 10% SDS-PAGE. Fraction purity and loading were controlled by immunoblotting for LAMP2 (a lysosomal marker). Red dashed arrows indicate immature cathepsins, while block solid arrows indicate mature cathepsins. (F) Co-localization of cathepsin B (CatB) and L (CatL) with lysotracker was observed in INS-1 cells treated daily with CBi and CLi in 30 mM glucose for 48 hr, as detected by immunofluorescence analysis. (G) Co-localization of CatB / CatL and protein disulfide isomerase (PDI) observed in INS-1 cells treated daily with CBi and CLi in 30 mM glucose for 48 hr. Nuclei were stained with Hoechst 33342 dye. The scale bar represents 5 μm.