Figure 1

Effects of MCF7 3-D spheroid formation on HIF-1 activation. A: Representative gel of Western blot analysis of HIF-1α performed on nuclear cellular extracts (see Materials and Methods for details) in MCF7 cell monolayers under normoxic conditions (Monolayer Normoxia), MCF7 cell monolayers under hypoxic conditions (Monolayer Hypoxia), MCF7 3-D spheroids (Spheroids), and Cos-7 cells stimulated with CoCl₂ (used as a positive control of HIF-1α expression). B: Representative gel of EMSA for detection of HIF-1α nuclear translocation. Monolayer Normoxia: MCF7 cell monolayers under normoxic conditions; Spheroids: MCF7 3-D spheroids; and Monolayer Hypoxia: MCF7 cell monolayers under hypoxic conditions as positive control (3% O₂ for 24 h). In each experiment one lane was loaded with bisdistilled water only (H₂O) in place of cellular extracts as negative control. This figure is representative of three similar experiments. C: Detection of HIF-1 activation by DNA binding ELISA in MCF7 cell monolayers under normoxic conditions (Monolayer Normoxia); MCF7 3-D spheroids (Spheroids); and MCF7 cell monolayers under hypoxic conditions (Monolayer Hypoxia). Cell monolayers and spheroids were incubated in the absence (white columns) or in the presence (hatched columns) of 5 μM YC-1 for 24 h. ***, P < 0.0001 versus Monolayer Normoxia without YC-1; °°°, P < 0.0001 versus Spheroids or Monolayer Hypoxia without YC-1. This figure is representative of three similar experiments, performed in duplicate.