Figure 4
Validation of the HCT116 DN model. HCT116 cells were stably transfected with an empty vector cassette (EV) or an hypoxia-inducible factor (HIF)-α dominant-negative construct (DN). Altered HIF-1 function was validated using an HIF reporter assay analysed in the presence or absence of hypoxia mimetic cobalt chloride (A) and by immunoblot analysis of the HIF-1 target glucose transporter-1 (GLUT-1) (B). At ∼500 μm, HCT116 wild type, EV or DN spheroids were treated with 100 μ M pimonidazole, fixed, sectioned and immunostained for GLUT-1 and pimonidazole adducts (C). Data are from a representative experiment showing the mean ±s.d. of triplicate samples (A); immunoblots (B) and immunostaining (C) are representative of three separate experiments. Scale bars indicate 100 μm.
