Figure 5

Effects of HIF-1α silencing on Pgp expression in MCF7 3-D spheroids and MCF7 hypoxic cells. Pgp protein levels were labeled on the surface of non-permeabilized cells and were quantified by FACS analysis as reported in Materials and Methods. A: Pgp protein levels were higher in MCF7 3-D spheroids (Spheroids), in MCF7 hypoxic cells (Monolayer Hypoxia), and in MCF7 cells transfected with MDR-1 gene (Monolayer Normoxia Pgp+) compared to Pgp protein levels in MCF7 control cells (Monolayer Normoxia). The transfection of cells with specific siRNA for HIF-1α (HIF-1α knockdown, KO) significantly decreased the Pgp surface expression in both MCF7 3-D spheroids and MCF7 hypoxic cells, while it had no effect in Pgp + cells. Silenced cells were indicated as HIF-1α KD 3-D Spheroids, HIF-1α KD Monolayer Hypoxia and HIF-1α KD Monolayer Normoxia Pgp+. These figure panels are representative of three similar experiments. B: The percentage of positive cells for Pgp in MCF7 cells cultured as monolayers under normoxic conditions (Monolayer Normoxia), 3-D spheroids (Spheroids), monolayers under hypoxic conditions (Monolayer Hypoxia), and transfected with MDR-1 gene (Monolayer Normoxia Pgp+). Cells and spheroids were transfected with 400 nM/L of HIF-1α (hatched columns) or transfected with vehicle alone (white columns). ***, P < 0.0001 versus Monolayer Normoxia transfected with vehicle alone; °°°, P < 0.0001 versus Spheroids or Monolayer Hypoxia transfected with vehicle alone.