Figure 3. [pIC]^PEI-triggered melanoma cell death is driven by autophagosome-autolysosome formation.

(A) Representative bright field (left and middle panels) and electron microscope (right panel) micrographs of SK-Mel-103 treated as indicated (30 h).

(B) Transformed wild-type or Atg5^-/- MEFs treated with low dose (0.2 μg/ml) of [pIC]^PEI or control. Cell death was estimated by trypan blue exclusion 48 h after treatment, and presented as means of three independent experiments.

(C) Confocal fluorescence images of SK-Mel-103 transduced by Cherry-GFP-LC3 to detect autophagosomes (red and green foci) and autolysosomes (red-only foci) formation after treatment with [pIC]^PEI, 25 nM Rapamycin (Rap) or solvent control.

(D) Inhibitory effect of 100 μM Bafilomycin (Bafil), 20 μM Chloroquine (Chlor) or 10μg/ml Pepstatin (PEP) on cell death estimated by trypan blue exclusion 20h after treatment with vehicle (white bars) or [pIC]^PEI (black bars). Data are indicated as means ± SEM of three independent experiments.

(E) Confocal fluorescence images of SK-Mel-103 cells stably transfected with eGFP-Rab5 WT and incubated with [pIC]^PEI labeled with Fluor Red. Shown is the internalization of [pIC]^PEI by the melanoma cells in the presence or absence of Chloroquine.

(F) Confocal visualization of lysosomal-dependent proteolysis upon cleavage and release of the fluorescent moiety of DQ-BSA (Green) in control or [pIC]^PEI-treated SK-Mel-103. Chloroquine is included to monitor DQ-BSA emission in cells with blocked lysosomal activity. Cells were simultaneously imaged in the presence of lysotracker red (LTR-red) to visualize the lysosomal compartment.

(G) The DQ-BSA-lysotracker colocalization was estimated in a minimum of 150 cells in two independent experiments, and it is expressed (as arbitrary fluorescence units) with respect to control treated cells. Error bars correspond to ± SEM of three independent experiments.