Figure 4.
Effect of hypoxia on hypoxia-inducible factor 1-alpha (HIF-1α), cyclooxygenase-2 (COX2) and epidermal growth factor receptor (EGFR)-related pathways and on epidermal-to-mesenchymal transition (EMT) markers following treatment with EGF with/without NS398. A: HT-29 cells were incubated for 24, 48 or 72 h under normoxic (NX) or hypoxic (HX) conditions. Nuclear and total cell lysates were analyzed by western blotting with specific antibodies for HIF-1α and COX2, respectively. B, C: HT-29 cells were treated for 24 h in the absence (control) or in the presence of EGF (100 ng/ml) and NS398 (75 μM), alone or in combination, under hypoxic conditions. Nuclear and total cell lysates were analyzed by western blotting with specific antibodies for HIF-1α, COX2, p-EGFR, phosphatidylinositol-3-kinase (PI3K), p-protein kinase B (p-AKT), phosphatase and tensin homolog (PTEN) and p-extracellular-signal-regulated kinases 1/2 (p-ERK1/2) (B) and for E-cadherin, N-cadherin and β-catenin (C). The optical density of the bands was determined by densitometry, normalized with respect to that of the corresponding β-actin band, and expressed as arbitrary units relative to the control (S.D. <10%). The blots shown are representative of three independent experiments.