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. 2016 Jun 9;7(32):51138–51149. doi: 10.18632/oncotarget.9939

Figure 3. VEGF-R2 targeting abolishes hypoxic stem-like melanoma cells.

Figure 3

(A) Hs294T cells were silenced with siRNAs against VEGF-R2 or with scramble siRNA as control. 48 hours after transfection, cells were treated with or without Etoposide (50 μM) under normoxic or hypoxic conditions for additional 24 hours. Melanosphere formation assay was performed and P1 spheres were quantified. (B) VEGF-R2 silencing was confirmed by Real Time PCR under normoxic or hypoxic condition at 24, 48, and 72 hours. Serum deprived cells in normoxic conditions were used as control. (C) Hs294T cells were serum starved for 24 hours and then incubated in the presence or absence of Etoposide (50 μM) and/or Bevacizumab (250 ng/ml) for 24 h. Melanosphere formation assay was then performed and P1 spheres were quantified. (D) Apoptotic cell death of Hs294T cells treated or not with Etoposide (50 μM) and/or VEGF-R2 siRNA, and/or Bevacizumab for 24 hours, was assessed by Annexin-V/propidium iodide staining. Phosphorylation of VEGF-R2 was analyzed on Hs294T cells treated or not with Bevacizumab (250 ng/ml) under normoxic or hypoxic condition. P values of ≤ 0.05 were considered statistically significant *p < 0.05, **p < 0.001, ***p < 0.0001, n = 3.