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. 2012 Oct 29;7(10):e38388. doi: 10.1371/journal.pone.0038388

Figure 2. ROS induce survival in hypoxic melanoma cells.

Figure 2

(A) 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). Percentage of apoptotic cells was measured with Annexin V staining versus propidium iodide-positive cells; *P<0,001 versus Etoposide treated control under hypoxia. (B) Cells were transfected with siRNA targeting Rieske Fe-S protein, serum-starved for 24 h and incubated in hypoxia and normoxia for additional 24 h. Percentage of apoptotic cells was measured by flow cytometry analysis of Annexin V versus propidium iodide-positive cells. *P<0,001 versus Etoposide treated control under hypoxia. (C) Cells were transfected with dominant negative RacN17 and after 24 h of serum starvation cells were exposed to 24 h of normoxia and hypoxia. Percentage of apoptotic cells was measured as in B. *P<0,001 versus Etoposide treated control under hypoxia. (D) 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. Hydrogen peroxide production was evaluated by DCDF-DA and normalized on protein content. *P<0,001 versus Etoposide treated control under hypoxia. (E) Hs29-4T cells were serum-starved for 24 h, then incubated under normoxia or hypoxia for 24 h in the presence of Apocynin in dose crescent stimulation, 1, 10 and 30 µM. Percentage of apoptotic cells was measured with Annexin V staining versus propidium iodide-positive cells; *P<0,001 versus Etoposide treated control under hypoxia.