Project description:Cell lines. HEK293 human embryonic kidney cells were obtained from ATCC (American Type Culture Collection, Manassas, VA) and were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Heat inactivated, Hyclone, Logan, Utah) and 1X antibiotics/glutamine (100 units/ml penicillin, 100ug/ml streptomycin, 292ug/ml L-Glutamine sulfate, all from Invitrogen Corp, Carlsbad, CA.), under either hypoxic (1% O2) or normoxic (21% O2) conditions at 37C in a tissue culture incubator. Treatment. Cells treated in hypoxia for 16 hours or transfected with HIF-1a (HIF-1amut or HIF2a) plasmid. At the end of treatment, cells were collected and washed. Microarray studies. Total RNA samples were extracted from HEK293T cells grown in 1% or 21% O2 overnight and from transfected cells. Total RNA (20 µg) from each sample was synthesized into double-stranded cDNA with reverse transcriptase (Fairplay labeling kit, Stratagen, La Jolla, CA) using an oligo d(T) primer. The double stranded cDNA from untreated cells was labeled with Cy3 monofunctional reactive dye and that from hypoxia-treated or transfected cells was labeled with Cy5 monofunctinal reactive dye. The probe was hybridized to a long oligo array (Hs-Operon V2-vB1.1px.gal) containing 20K human transcripts (NCI Microarray Facility, Advanced Technology Center, Gaithersburg, MD) overnight at 42C . The slides were washed and spin-dried. Microarray slides were scanned with a GenePix 4000 microarray scanner (Axon Instruments, Union City, CA.). The microarray images were analyzed with GenePix 3.0 software and data analysis was performed with the MicroArray Database (mAdb) system hosted by the Center for Information Technology and Center for Cancer Research at the NCI. Keywords: other
Project description:HIF-1A and HIF-2A regulate both overlapping and unique target genes in response to hypoxia. In this dataset, we identify specific HIF-1A and HIF-2A target genes in glioblastoma cells. 12 samples were analysed comprising 4 experimental conditions (normoxia scr, hypoxia scr, hypoxia siHIF1, hypoxia siHIF2) in triplicate. We made pairwise comparisons between the averages of each triplicate set to normoxia scr using the Partek suite.
Project description:HIF-1A and HIF-2A regulate both overlapping and unique target genes in response to hypoxia. In this dataset, we identify specific HIF-1A and HIF-2A target genes in glioblastoma cells.
Project description:HIF-1a and HIF-2a are expressed at high levels in mesenchymal progenitors compared to more committed mesenchymal cells and hematopoietic cells. HIF-factors could therefore have a role in the regulation the biology of mesenchymal progenitors and their functions, like the non cell-autonomous maintenance of hematopoietic progenitors. We used microarrays to detail the global program of gene expression regulated by HIF-1a or HIF-2a in mesenchymal progenitors Mesenchymal progenitors were FACS-sorted and cultured in low oxygen concentration for few days. Once cells started to form CFU-F colonies, we transduced them with shRNAs targeting specifically HIF-1a or HIF-2a. Four days after transduction, cells were collected and RNA extracted for microarray analysis.
Project description:HIF-1a and HIF-2a are expressed at high levels in mesenchymal progenitors compared to more committed mesenchymal cells and hematopoietic cells. HIF-factors could therefore have a role in the regulation the biology of mesenchymal progenitors and their functions, like the non cell-autonomous maintenance of hematopoietic progenitors. We used microarrays to detail the global program of gene expression regulated by HIF-1a or HIF-2a in mesenchymal progenitors
