Fig. 3.

Clusterin (CLU) protects pancreatic β-cells against lipotoxicity-induced apoptosis. Mouse insulinoma 6 (MIN6) cells were incubated with 0.5 mM palmitate (PA) in the presence or absence of 10 μg/L CLU for 24 hours. (A, B) Autophagy-inducing or -related proteins, including light chain 3 (LC3), autophagy-related 3 (Atg3), p62, beclin-1, Unc-51 like autophagy activating kinase (ULK)1, phosphor-mammalian target of rapamycin (mTOR), and β-actin were measured by Western blotting, and the respective ratios of LC3-II, Atg3, p62, and beclin-1 to LC3-I and β-actin were described (n=3–4 in each treatment group). (C, D) Terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) staining was performed to detect apoptosis in MIN6 cells, and TUNEL-positive signals are indicated by red spots. Apoptotic cells are represented by arrowheads that indicate red spots in the nuclei. The nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI). Scale bars=20 μm. The number of apoptotic cells was counted and described by a ratio to 100 cells (n=5–6). (E, F) The protein level of cleaved caspase-3 (c-casp3), a marker of apoptosis, was examined by Western blotting, and the level of c-casp3 was normalized to total caspase-3 (t-casp3). All values are expressed as mean±standard error of the mean (n=3–4). ^aP<0.05 and ^bP<0.01 compared to vehicle (VEH); ^cP<0.05 compared to PA.