Hypoxic Activation of K-Ras Figure 6. Induction of VEGF under hypoxic conditions is suppressed by inhibition of c-Src. A and C, Relative mRNA levels of VEGF, as evaluated by quantitative RT-PCR, in Caco2 and HT29 cells pretreated with 10 mM PP2 (A) or transiently transfected with a Src-specific siRNA construct (C), and exposed to normoxia or hypoxia for 24 hours. The data are expressed as fold change as compared to control cells in normoxia, normalized to 1. *, P,0.05. B and D, VEGF luciferase reporter assays of Caco2 and HT29 cells, pretreated with 10 mM PP2 (B) or transiently transfected with a Src-specific siRNA construct (D), and exposed to normoxia or hypoxia for 24 hours. The results are from three independent experiments carried out in duplicate and are presented as fold change as compared with control cells in normoxia normalized to 1. Data are shown as mean 6 SD. *, P,0.05. **, P,0.01. doi:10.1371/journal.pone.0010966.g006 siRNA analysis siRNA specific against K-ras (Ambion ID: 6752 and ID: 120702; Austin, TX) and c-Src (on-target plus SMART pool human c-Src, Dharmacon Inc. ID: L-003175-00 and Ambion ID: s13414; Lafayette, CO) were utilized. A control siRNA that does not correspond to any known human gene was also utilized. Cells were plated to reach 30-50% confluence on the day of transfection and 20 nM siRNA duplexes were introduced using Lipofectamine RNAiMAX (Invitrogen). Establishment of stable cells Lentivirus vector pCSGW GFP-LC3 was kindly provided by Dr. Ramnik Xavier (Massachusetts General Hospital, Boston, MA, USA), and the GFP-LC3 fragment was excised with BamHI and NotI. The human K-ras cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using mRNA from SW480 cells that have homozygous mutation at codon 12 of KRAS gene (G12V). pCSGW K-ras V12 or empty vector were introduced with packaging plasmid pCMV-dR8.91 and envelope plasmid pMD2.G into HEK293T cells by transfection with the Fugene6 regent (Roche, Indianapolis, IN). The culture media containing lentivirus was harvested twice every 24 hours and filtered, and polybrene was added at 8 mg/mL. Caco2 cells were infected by culturing with the virus containing medium for 48 hours. Transfection Caco2 cells were transfected with empty vector (pEVX) or constitutively active c-Src plasmid (Src Y527F) using Lipofecta- mine 2000 (Invitrogen). Forty-eight hours after transfection, cells were harvested for a Ras activation assay. Plasmids containing c- Src gene mutants and pEVX vector were kind gifts from Dr. David Shalloway and Dr. Michael Botchan, respectively (Ad- dgene, Cambridge, MA). Reporter assays Luciferase reporter assays were performed in 24-well tissue culture plates. Cell were cotransfected with 0.6 mg of a 2.3 kb VEGF-luciferase reporter construct and 2 ng of pRL-CMV (Promega, Madison, WI) using Fugene6 (Roche) [17]. Twenty- four hours later the media was switched to UltraCulture and cells were exposed to normoxia or hypoxia for additional 24 hours. Luciferase activity was measured with a dual luciferase reporter assay system (Promega). ELISA Culture media and cellular extracts were collected after 24 hours of normoxic or hypoxic conditions. VEGF protein levels were assayed using a human VEGF-specific ELISA Kit (Quanti- kine; R&D Systems, Minneapolis, MN) and measured by a microplate reader set to 450 nm. Western blotting Protein lysates were harvested from cells incubated in normoxia or hypoxia for the indicated periods. Cells were lysed in chilled lysis buffer (Cell Signaling) with Protease inhibitor cocktail (Roche). Lysates (18–50 mg) were resolved on a Bis-Tris poly- acrylamide or Tris-Acetate gel (Invitrogen) and transferred onto a polyvinylidene fluoride (PDVF) membrane (Millipore, Bedford, MA). Membranes were blocked in 5% non-fat milk containing 0.1% Tween 20 (TBS-T) for 1 hour at room temperature and incubated overnight with relevant antibodies at 4uC. Secondary antibodies coupled to horseradish peroxidise were visualized using the Western Lighting Chemiluminescence Reagent Plus (Perki- nElmer Life Sciences, Boston, MA). Ras activation assay The level of active Raf-bound Ras was assessed utilizing a Ras activation assay kit (Upstate, Lake Placid, NY). Cells were cultured to 70%–80% confluence in a 15 cm dish and switched to DMEM with 2% FBS before incubation in normoxia or hypoxia for 4 hours. Two milligrams of each cell extract was mixed with 10 mL of the Ras Assay Reagent (Raf-1 RBD agarose slurry) and incubated at 4uC for 45 minutes. The agarose beads were resuspended in 40 mL of 2x Laemmli reducing sample buffer and boiled. Twenty microliters of lysates were separated by SDS- PAGE and transferred to a PVDF membrane. Active Ras was detected using a Ras antibody (kit component) or an isoform specific antibody to K-ras or N-ras (both Santa Cruz). Annexin V-PI staining for fluorescence microscopy An apoptosis assay was performed using ApoAlert Annexin V kit (Clontech, Palo Alto, CA). Colo320DM cells grown on sterile coverslips were transfected with control or K-ras siRNA, incubated in hypoxia for 48 hours, and then incubated with Annexin V-FITC and Propidium Iodide (PI) in the dark for 10 minutes at room temperature. Coverslips were inverted on glass slides and cells were visualized with an Olympus AX70 microscope (Olympus, Canter Valley, PA). Determination of apoptosis by flow cytometry Caco2 cells were transfected with K-ras or control siRNA 24 hours before incubation in hypoxia. Cells which were irradiated under UV before incubation with Cycloheximide (10 mg/mL) and TNFa (100 ng/mL) in normoxia served as a positive control. Cells were trypsinized and resuspended in 200 mL binding buffer (ApoAlert Annexin V kit) containing 5 mL of annexin V-FITC stock and 10 mL of a 50 mg/mL solution of propidium iodide (PI). After incubation for 15 minutes at room temperature, the samples were analyzed by FACSCalibur (BD Bioscience, Bedford, MA) using Flowjo software. For each measurement, 100,000 cells were collected. Statistical analysis Statistical differences were analyzed by the Student’s t test, and P values,0.05 were considered statistically significant. Supporting Information Figure S1 N-ras is not activated by hypoxia. Found at: doi:10.1371/journal.pone.0010966.s001 (0.15 DOC) MB Figure S2 Over-expression of c-Src increases K-ras activity. PLoS ONE | www.plosone.org 10 June 2010 | Volume 5 | Issue 6 | e10966