normalization data transformation protocol
Raw data files from Feature Extraction were imported into R with LIMMA (Smyth, 2004, Statistical applications in Genetics and molecular biology, vol3, N1, article3), an R package from the Bioconductor project, and processed as follow: gMedianSignal and rMedianSignal data were imported, controls probes were systematically removed, and flagged probes (gIsSaturated, gIsFeatpopnOL, gIsFeatNonUnifOL, rIsSaturated, rIsFeatpopnOL, rIsFeatNonUnifOL) were set to NA. Intra-array normalization was performed by a loess normalization, followed by a quantile normalization of both Cy3 and Cy5 channel.
array scanning and feature extraction protocol
After washing in acetonitrile, slides were scanned by using an Agilent G2565 C DNA microarray scanner with defaults parameters (100 PMT, 3 um resolution, at 20C in free ozone concentration environment. Microarray images were analysed by using Feature Extraction software version (10.7.3.1) from Agilent technologies. Defaults settings were used.
nucleic acid hybridization to array protocol
Hybridization were then performed on microarray using 825 ng of each linearly amplified cRNA labelled Cy3 or Cy5 sample, following the manufacturer protocol (Agilent SureHyb Chamber; 1650ng of labeled extract; duration of hybridization of 17 hours; 40 uL per array; Temperature of 65C).
nucleic acid labeling protocol
Probe synthesis and labeling were performed by using the two-color Agilent labeling kit (Low Input Quick Amp Labeling Kit 5190-2306)
Gene silencing of HIF-2a and ITPR1 was performed using predesigned sequence-specific small interfering RNA purchased from SIGMA Aldrich. Briefly, 8 X 106 cells were electroporated twice in 48 h in serum-free medium with 20 mM siRNA in an EasyJect Plus electroporation system (EquiBio; 260 V, 450mF).
nucleic acid extraction protocol
Total RNA was extracted using TRIzol solution (Invitrogen).
786-0, PRC3 and WT7 are renal carcinoma cells obtained from Dr. Kaelin WG Jr. 786-0 cells were maintained in DMEM/F12 medium supplemented with 10% heat-inactivated FCS, 1% penicillin-streptomycin, and 1 mM sodium pyruvate (Life Technologies, Cergy Pontoise, France). WT7 and PRC3 cells were derived from 786-O by stable transfection with pRC-HAVHL or the empty vector respectively and maintained in 786-O medium added with 1 mg/ml of neomycin as selecting agent. NK HD cells were purified from healthy donor peripheral blood mononuclear cells (PBMC) using CD56 positive selection (STEMCELL technologies). They were cultured in RPMI supplemented with 10% human serum, 1% penicillin-streptomycin, 1 mM sodium pyruvate, and IL2 (300U/ml, Institut Gustave Roussy). NKL and NK-92 natural killer cell lines were maintained in RPMI supplemented with 10% heat-inactivated FCS, 1% penicillin-streptomycin, and 1mM sodium pyruvate (Life Technologies, Cergy Pontoise, France). RENCA murine renal carcinoma cell line was originally obtained from a tumor that arose spontaneously in the kidney of BALB/c mice (25). It was maintained in RPMI medium supplemented with 10% heat-inactivated FCS, 1% penicillin-streptomycin, and 1 mM sodium pyruvate (Life Technologies, Cergy Pontoise, France).