Figure 5.

Late-stage autophagy blockage is involved in SOR + STV-induced cell death of melanoma cells. (A) Autophagy was evaluated by western blotting analysis, by measuring LC3 conversion and p62 degradation in CHL-1 and SK Mel 28 cells exposed 6 h to EBSS (STV), SOR [10 mM] or SOR + STV, in presence or absence of the autophagic inhibitor chloroquine (CQ 25 mM). Tubulin was used as loading control (Images are representative of three independent experiments). (B) Cell viability of CHL-1 and SK Mel 28 was also performed in the same experimental conditions reported in A. (Histograms represents mean ± SD; n = 3) * p < 0.005 compared to control cells. # p < 0.0001 absence vs. presence of CQ. (C) ATG5 expression was abrogated in both CHL-1 and SK Mel 28 by transducing cells with lentiviral particles carrying a specific shRNA sequence for Atg5 (shAtg5) or a scrambled sequence (scramble). Atg5 protein levels were evaluated by western blotting analysis (left panel) and a densitometric analysis (Atg5 normalized by GAPDH) was also performed (right panel). GAPDH was used as loading control. (Images are representative of three independent experiments; histograms represent mean ± SD, n = 3) * p < 0.0001 compared to not transduced cells (Φ); # p < 0.0001 compared to scramble cells. (D) Cell viability was evaluated by flow cytometry in both control cells and shAtg5 cells under STV, SOR, or combined treatment (SOR + STV), at 6 h post treatment. (Histograms represent mean ± SD; n = 3) * p < 0.0001 compared to untreated cells (CTR).