Fig. 6

Sirt1 knockdown blocked the upregulation of melatonin on autophagy and mitophagy. A Expression levels of SIRT1 and AMPK in TM3 cells were detected by Western blot and their gray values were analyzed; B The expression levels of autophagy-related proteins Beclin-1, Atg7, Atg5 and LC3 in TM3 cells were detected by Western blot, and the gray values of protein bands were statistically analyzed; C-F. Mitophagy in TM3 cells was marked by COXIV and LC3 antibody co-staining. The fluorescence intensity and the percentage of co-stained cells (counting randomly 150 cells in six visual fields) were statistically analyzed. G-J Immunofluorescent co-staining of BNIP3L and LC3 antibodies in TM3 cells. The fluorescence intensity (randomly counting 100 cells in 10 visual fields) and the percentage of co-stained particles in the cells (totally 30 cells) were statistically analyzed. K The protein level of BNIP3L in TM3 cells was detected by Western blot, and the gray values of the protein bands were analyzed statistically. NG, normal glucose control group; HG, high glucose treatment group; HG + MT, high glucose and melatonin treatment group; siSirt1, HG + MT + siSirt1 group. A-B, Data are expressed as fold change relative to NG. *, P < 0.05; #, P < 0.01. Bar: C, 100 μm; G, 10 μm