Figure 3

APE induces beclin-independent autophagy in MDA-MB-231 cells. (a) Representative images of LysoTracker Red staining of MDA-MB-231 cells treated or not (control) with APE 300 μM EqC for 24 h and analyzed by fluorescence microscopy. Chloroquine is used as positive control. (b) MDA-MB-231 cells were treated with APE 100 and 300 μM EqC at indicated times and then flow cytometry analysis was performed. At least 2 × 10⁴ events were acquired in log mode. For the quantitative evaluation of LTR, FlowJo software was used to calculate median fluorescence intensities (MFI) by the formula (MFI-treated/MFI-control). The hystogram represents the values of MFI of LTR analyzed by flow cytometry, as a percentage of the control (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control cells). Analysis was carried out by triplicate determination on at least three separate experiments. (c) MDA-MB-231 cells were treated with APE 100 and 300 μM EqC for 48 h. The protein content of LC3B, p62, and beclin-1 was analyzed by western blotting. The graph shows the densitometric intensity of LC3B II/I bands ratio. The intensities of signals were expressed as arbitrary units (*P < 0.05 versus control cells). α-tubulin was used as loading control. The full-length blots are included in the supplementary information (Fig. S4).