Figure 3.

P2Et treatment induces autophagy in B16F10 melanoma cells. Cells were cultures in complete media for 48 h and then treated with P2Et 1/2 IC50, Dx IC50, Met 500 μM or with the negative controls (EtOH and DMSO, vehicles). Autophagy induction was evaluated by two different methods. Images were acquired in a Olympus FV1000 microscopy with a 60 × PlanAPO objective. (a) Cells were cultured for 24 h with P2Et, Met (autophagy inducer, positive control), Dx or negative control, then stained with 0.05 mM MDC (green) in complete media for 1 h a 37 °C washed and actin filaments stained with phalloidin Alexa Fluor 594 (red) (n=3). (b) Cells were cultured for 12 h with P2Et, Dx (autophagy and ICD inducer, positive control) or in control conditions. The last 3 h of the culture (10 nM) bafilomycn A was added to the media as an inhibitor of autophagic flux. Cells were stained the rabbit anti-mouse LC3 polyclonal primary antibody and reveled with a rabbit polyclonal Alexa Fluor 488 secondary antibody (green). DAPI was used to stain the nuclei (blue). (c) Quantitative data (Mean±S.E.M., n=2) is reported ****P<0,0001; **P<0,01 (n=3). (d) Cells were treated with P2Et or with the negative control (EtOH) for 24 h. Then, lysates were evaluated for the presence of p62 protein by western-blot