Fig. 9

Autophagy modulation after Pyr and MBP treatment. Autophagy was evaluated utilizing two different assays: a cationic amphiphilic dye that selectively labels autophagic vacuoles (Cyto ID autophagy detection kit) by flow cytometric analysis (Fig. 9a-b ) and the expression of LC3I/II by WB analysis (Fig. 9c-d ). Mel501 melanoma cells were treated with Pyr (8 μg/ml), and MBP (0.8 μg/ml) for 48 h. The experiments were performed in the presence or absence of the late autophagy inhibitor CQ (10 μM) for an overnight treatment before the end of the experiment. As shown by fluorescence histograms of Cyto-ID Green autophagy dye (Fig. 9a-b ), Pyr treatment was able to induce a significant increase of autophagic vacuoles. Note, the induction of autophagy can be better visualized when the removal of these vesicles is prevented by CQ. Treatment with MBP did not induce significant autophagy and (as expected) CQ treatment (MBP/CQ) did not induce any significant effect. Figure 9a , Columns, mean values of three independent experiments; bars, SD. *, P < 0.05; significance compared with untreated cells. #, P < 0.05; ##, P < 0.01; significance compared with CQ treated cells. In Fig. 9b the data of a representative experiment were shown. The data were confirmed by western blot of LC3-II turn-over (Fig. 9d ) in the presence or absence of CQ. The intensities of bands of LC3II were quantified by densitometric analysis (Fig. 9c ). The values were normalized to Tubulin and expressed as arbitrary units (AU). Columns, mean values of three independent experiments; bars, SD. *, P < 0.05; significance compared with untreated cells. #, P < 0.05; ##, P < 0.01; significance compared with CQ treated cells. As Pyr was able to induce both apoptosis and autophagy, we have also evaluated Pyr-induced apoptosis in the presence of CQ. As shown in Fig. 9e , CQ significantly enhanced Pyr-induced apoptosis (about 2-fold) while MBP/CQ treatment did not induce any significant effect. Columns, mean values of three independent experiments; bars, SD. *, P < 0.05; significance compared with untreated cells. #, P < 0.05; ##, P < 0.01; significance compared with CQ treated cells. In Fig. 9f a representative experiment was shown