Fig. 8

The detection of autophagy by acridine orange staining (AO). a–n Fluorescent microscope was used to visualize the acidic vesicular organelles (AVOs; red fluorescence) as well as the cytoplasm and nucleus (green fluorescence) after the vital staining of the cells with AO, as indicated in Materials and methods. a–h The A549 cells were treated with 10 µM fisetin (FIS) and/or 0.1 µM paclitaxel (PTX) for 24 h or left untreated (control, CTRL). i, j As negative control, bafilomycin A1 (Baf A1; 100 nM) was added to the cells for a period of 4 h, followed by washing with PBS and subsequent incubation with FIS and PTX for 24 h. k–n In another set of experiment, the cells were treated with the combination of 10 µM fisetin and 0.1 µM paclitaxel for 24 h, followed by post-treatment incubation in a drug-free medium for the next 24 or 48 h. Note the increased amount of AVOs after the treatment with FIS and/or PTX (c, e, g). Arrowheads indicate: (I) the giant multinucleated cells filled with numerous AVOs. Bar 50 µm. o The measurement of the red fluorescence of AO using image-based cytometer. Asterisks represent statistically significant differences from control cells, and symbol ^{^} indicates statistically significant differences compared to the treatment with FIS plus PTX (p < 0.05; Mann–Whitney U test). Symbols ^$ and ^# indicate statistically significant differences compared with FIS or PTX treatment alone, respectively (p < 0.05; Mann–Whitney U test). Data are representative of three separate experiments and presented as mean and standard deviation