Figure 4

Betulinaldehyde promoted the autophagy flux in A549 cells. (A) A549 cells were treated with different concentrations of betulinaldehyde for 24 h before the autophagy flux was analyzed via a confocal laser scanning microscope using mCherry-GFP-LC3B. (B) 3-MA was also used to determine the effects of 20-μM betulinaldehyde on the autophagy flux in A549 cells. (C,D) Quantification of LC3B positive autolysomomes or autophagosomes in A549/mCherry-GFP-LC3B cells. (E) Expressions of SQSTM1 and LC3-II were tested at the protein level by western blotting. The statistical analyses of the (F) SQSTM1 levels and LC3-II levels are included. Cropped images are displayed, uncropped blots are displayed in Supplementary Fig. 3. The images were collected from different parts of the same gel with same loading amounts. The blot gels shown are representatives of three different experiments (n = 3). All values are expressed as the mean ± SD (n = 3). Comparisons among multiple groups were performed using one-way analysis of variance. The differences between control and other groups were analyzed using LSD multiple comparison test. *P < 0.05, **P < 0.01 versus control group, ^#P < 0.05, ^##P < 0.01 versus Bet group (Bet = betulinaldehyde, 3-MA = 3-methyladenine). Scale bar, 20 μm.