Fig. 3.

Ethanol extracts of Chrysanthemum zawadskii Herbich (ECZ) induced autophagy in mouse colon cancer CT-26 cells. a Detection of acidic vesicular organelles (AVOs). b Representive graph of autophagic celss. Cells were treated with various concentrations (100–300 μg/ml) of ECZ for 24 h and stained with 1 μM acridine orange (AO) at 37 °C in the dark for 20 min and then analyzed by flow cytometry. c Expression of LC3B and p62/SQSTM1. d Relative fold change of LC3B-II. e Relative fold change of p62/SQSTM1. Total cell lysates were analyzed by Western blotting. f Pretreatment with 3-MA reversed the ECZ-induced increase in autophagy. g Effect of 3-MA on LC3B and p62/SQSTM1 expression. h Relative fold change of LC3B-II. (I) Relative fold change of p62/SQSTM1. Cells were pretreated with 1 mM 3-MA prior to 1 h and AO-stained cells were evaluated by flow cytometry. Total lysates were subjected to SDS-PAGE for Western blot analysis. Data are presented as the mean ± standard deviation for at least three independent experiments. **p < 0.01, ***p < 0.001 vs. the control group; #p < 0.05, ^##p < 0.01 vs. the ECZ treated group