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. 2020 Apr 6;16(11):1901–1916. doi: 10.7150/ijbs.44343

Figure 5.

Figure 5

Effect of MHY2245 or doxorubicin (DOX) on the autophagic cell death pathway in SKOV3 cells. (A) Cells were treated with vehicle control, MHY2245 (0.03, 0.1 or 0.3 µM) or DOX (1 µM) for 48 h and the expression of autophagy-related proteins was analyzed by Western blot. β-Actin was used as a loading control. (B) Representative histogram showing the expression levels were quantified using Image J software compared to β-actin. Data expressed as mean ± SD of triplicate experiments. *p < 0.05 and **p < 0.01 and ***p < 0.01versus the control group. (C) Cells were treated with vehicle control, MHY2245 (0.03, 0.1 or 0.3 µM) or DOX (1 µM) for 48 h. After fixed, cells were incubated with MDC (0.05 mM) for 10 min at 37 °C and then washed four times with PBS pH 7.4. Cells were immediately analyzed by fluorescence microscopy using an confocal laser scanning microscope. (D) Acridine orange staining was used to detect the formation of autophagic vacuoles in SKOV3 cells treated with MHY2245 or DOX for 48 h. The cytoplasm and nucleolus fluoresce green, whereas the acidic compartments fluoresce bright red or orange-red colors. Images were observed using a confocal laser scanning microscope (LSM 510, Magnification × 400). Scale bar, 10 µm. (E) The acidic vacuoles were determined using flow cytometry.