Inhibition of cathepsins B and L leads to LC3 accumulation.

(A) Activity of cathepsins B and L in 30 mM glucose with E64d (E, 20 μg/mL), cathepsin B inhibitor (CBi, 20 μM) or cathepsin L inhibitor (CLi, 20 μM) was measured at 24 hr, 48 hr and 72 hr (n = 9). Statistical significance is represented as *** p <0.001 compared with 30 mM glucose. (B) INS-1 cells cultured in 30 mM glucose for 24 or 48 hr were treated daily with E, P, CBi, and CLi. LC3-II accumulation was assessed by immunoblot analysis. (C) Islets from SD rat cultured in 30 mM glucose and treated with CBi, and CLi. Level of LC3 was measured by immunoblot assay. (D) GFP-LC3/INS-1 stable cells were treated daily with E, P, CBi, and CLiin in 30 mM glucose for 48 hr. The graph indicates the number of GFP-LC3 puncta counted among GFP positive cells (n = 5). *** p <0.001 compared with 30 mM glucose. The scale bar represents 5 μm.