Hypoxic Activation of K-Ras Figure 3. Activation of Akt by hypoxia is downstream of c-Src and K-ras. A, Protein extracts from Caco2 and HT29 cells grown in normoxic or hypoxic conditions for the indicated times were subjected to immunoblotting for phospho-Akt and phospho-ERK1/2. The blots were then stripped and reprobed with antibodies against total Akt and total ERK. b-actin was used as a loading control. Densitometry values for p-Akt are expressed as fold change compared with control values normalized to 1. B, Caco2 cells were incubated for 1 hour with PP2 at the concentrations indicated, before transfer to normoxic or hypoxic conditions for 12 hours. Western blotting was performed to determine the levels of phospho-Akt, total Akt, phospho- Src416, and total Src. b-actin was used as a loading control. Densitometry values are expressed as fold change compared with control values normalized to 1. C, Caco2 cells were transfected with control siRNA, K-ras siRNA or c-Src siRNA oligos (each at 20 nM) before exposure to normoxic (N) or hypoxic (H) conditions for 12 hours. Immunoblotting with the indicated antibodies was then performed. Densitometry values for p-Akt are expressed as fold change compared with control values normalized to 1. D, Caco2 cells stably overexpressing K-rasV12 (Caco2pCSGWK-ras V12) were transfected with c-Src siRNA oligos. Forty-eight hours later, cells were incubated in hypoxia or normoxia for 12 hours and Western blotting with the indicated antibodies was then performed. Densitometry values for p-AKT are expressed as fold change compared with control values normalized to 1. doi:10.1371/journal.pone.0010966.g003 Hypoxic regulation of VEGF by K-ras Hypoxia is also a potent stimulus for angiogenesis through the induction of VEGF. We previously demonstrated that hypoxia and oncogenic K-ras can synergistically up-regulate VEGF [2]. While such studies provide invaluable insights into oncogenic K-ras function, they do not provide insights into mechanisms that colon cancer cells with a wild-type KRAS may utilize. We therefore inhibited the endogenous wild-type KRAS in Caco2 cells with PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e10966