Figure 5.

5,6-ECs induce an autophagy-mediated cell death. (A) U266 cells were treated with vehicle or 5,6 α/β-EC (20, 40 µg/mL) for 20 h. Cells were then stained with an autophagosome specific fluorescent probe and analyzed with an Olympus BX53 fluorescent microscope (× 40, magnification) (B) p62 expression was analyzed by IF in EtOH- and 5,6-ECs-treated cells. We used a primary Ab against p62, and a goat Alexa Fluor 488-conjugated anti-rabbit IgG as secondary Ab. Slides were counterstained with DAPI and analyzed with a confocal microscope (Fluoview FV100, Olympus, CA, USA) (× 180, magnification). (C) U266 cells were untreated (U), vehicle-treated (E) or with 5,6 α-EC (40 µg/mL) or 5,6 β-EC (20 or 40 µg/mL). Within 24 h, whole-cell proteins were extracted, separated by SDS-PAGE, and transferred onto membranes then incubated with anti-LC3B or anti-β-actin (as a control) antibodies. Protein levels were estimated by densitometry and collected data from three independent experiments were presented in histograms (means ± SD). (D). The modification of autophagic flux was evaluated by flow cytometry. U266 cells were seeded into 24-well plates for 24 h. The cells were treated with 5,6 α-EC or 5,6 β-EC alone or in combination with 5 µM rapamycin or 10 µM 3-MA for 24 h. The cells were stained with PI and PI positive cells were recorded. At least 10⁴ events were gated. The experiment has been done once with triplicate samples. (E) U266 cells were pre-treated with BafA1 (50 nM) for 4 h and then treated with 5,6 β-EC (40 μg/mL). The cells were stained with annexin V/IP as described before and sorted. Cytometry profiles were presented together with the percentage of cells within each quadrant. The percentage of annexin V+ cells in each culture condition is presented in the histogram. * p <0.05; ** p < 0.01; *** p < 0.001; ns, not significant with the t-test.