Figure 1. Hypoxia induces cell survival in melanoma cells.

Hs29-4T cells were serum-starved for 24 h and then incubated under normoxia or hypoxia in the absence or presence of etoposide (50 µM) for additional 24 h. Different parameter revealing cell death or cell survival were examined. (A) Percentage of apoptotic cells was measured with Annexin V staining versus propidium iodide-positive cells; averages SD values are shown in the bar graph. *P<0,001 versus etoposide-treated under hypoxia. (B) Cells were stained with specific antibody against caspase 3. Cells positive to caspase 3 staining were revealed by flow cytometric analysis; averages ±SD values are shown in the bar graph. *P<0,001 versus etoposide-treated under hypoxia. (C) Living cells were stained with TMRN and mitochondrial fluorescence (excitation 543 nm, emission 590 nm) was quantified by fluorimetric assay (FluoroSkan) and normalized on protein content; averages ±SD values are shown in the bar graph. *P<0,001 versus etoposide-treated under hypoxia. (D) After cells lysis p-Akt and p-53 proteins accumulation were assessed with western blotting. β-Actin is shown as loading control. Image quantification reports fold increase in protein expression. (E) Hs29-4T cells were serum-starved for 24 h, then incubated under normoxia or hypoxia for 24 h in the absence or presence of specific inhibitors of the various form of ROS, namely DPI (5 µM) and rotenone (1 µM). Hydrogen peroxide production was evaluated by DCDF-DA and normalized on protein content. *P<0,001 versus untreated control under hypoxia.