FIGURE 6.

MA was diminished in melanomas with increased LAMP-2C. (A–C) DM331 or SLM2-Mel cells were incubated overnight with ±20 μM CQ, an inhibitor of lysosome acidification, to monitor autophagosome formation and turnover. To measure MA flux, the conversion of LC3I to LC3II was detected and normalized to cellular actin levels. MA flux was quantified to detect changes in cellular LC3II levels ± CQ using the equation MA flux = LC3II/actin in CQ treated cells – LC3II/actin in control cells. The relative levels of LC3I/actin in each cell line grown without CQ provides a measure of basal LC3I protein accumulation prior to its enzymatic conversion to LC3II during MA. (D) DM331 pCMV and DM331 2C myc cells were incubated with media ± serum, stained with CYTO-ID, and MA monitored by flow cytometry. The geometric mean was set equal to one for DM331 pCMV cells for relative comparison to the geometric mean in DM331 2C myc cells. Data were analyzed by two-way ANOVA or by two-tailed, unpaired Student’s t-test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 (n = 3).