Pegfp

RIP140-expressing vectors (pEFcmyc-RIP140 [47 (link)], pEGFP-RIP140[48 (link)]) and control vectors, pEGFP (Clontech), are described elsewhere. HA-HIF1alpha-pcDNA3 (Addgene plasmid # 18949) and HA-HIF2alpha-pcDNA3 (Addgene plasmid # 18950) plasmids were gifts from Dr Kaelin [49 (link)]. The GLUT3-Luc reporter gene was a gifts from Dr. Yuan [50 (link)]. 2-Deoxy-d-glucose (2DG, D6134)), 6-aminonicotinamide (6AN; # A68203), 3-bromopyruvate (BrP; #16490), MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (98%, CAS 298-93-1), deferoxamine mesylate salt (D9533), di(N-succinimidyl)glutarate (80424), crystal violet (C0775), anti-Mouse IgG-FITC antibody (F6257), monoclonal Anti-β-actin-peroxidase antibody (A3854) were purchased from Sigma-Aldrich (Merck, Darmstadt, Germany). Puromycin (ant-pr-1) was purchased from Invivogen (France). The ATP Determination Kit (10700345) was from Fisher Scientific (France). Deoxy-d-glucose, 2-[1,2-3H (N)] (NET328A250UC) was from Perkin Elmer (France). GLUT3 shRNA (m) (sc-41219-V) were purchased from Santa Cruz Biotechnology (Dallas, USA). Rabbit polyclonal to RIP140 (ab42126) was from Abcam (Cambridge, UK). BrdU Hu-purified-clone B44 (#347580) was from Becton Dickinson (France). Ambion™ Silencer™ Pre-Designed siRNA specific of human GLUT3 (SLC2A3) was purchased from Fisher Scientific (#10446914, Illkirch, France).

RT-PCR was used for cloning of PRG3 from rat, mouse and human mRNA samples. For sequence alignments and homology searches of PRGs we utilized the www.ncbi.nlm.nih.gov database and A Plasmid editor software (ApE; MW Davis, Utah, USA). Trans-membrane domains have been predicted using the Kyte Doolittle algorithm and all orthologous sequences of PRG3 (human, mouse and rat) are deposited at the NCBI database (Human PRG3 GenBank accession no. AY304516; Rattus norvegicus PRG3 GenBank accession no. AY299399; Mus musculus PRG3 GenBank Accession no. AY345342). We amplified the fragments by PCR and cloned the resulting amplicons into the pEGFP (Takara, Heidelberg, Germany), and pCLEG (Stefan Schumacher, Ulm, Germany) vectors. C-terminal domain of PRG3 was cloned respectively. According to the criteria of Naito et al [51 (link)] two short interfering RasGRF1 RNAs were designed. Cloning of the synthetic oligonucleotides into the pSuperRFP vector (modified pS-GFPneo; OligoEngine, Seattle, USA) was performed by digesting the empty vector with Bgl2 and EcoR1 according to the manufacturer's instruction.

Reverse transcription-polymerase chain reaction was applied for full length cloning of xCT from rat mRNA samples. For sequence alignments and homology searches of xCT we utilized the www.ncbi.nlm.nih.gov database and A-Plasmid editor software (ApE; MW Davis, Utah, USA). The sequences of rat xCT are deposited at the NCBI database (GenBank accession no. NM001107673). For construct cloning we cloned fragments by PCR and inserted the resulting amplicons into the pEGFP (Takara, Heidelberg, Germany) vector. According to the critera of Ui-Tei et al. [22] (link) three 19-mer short interfering RNAs were chosen for RNA interference with rat xCT transcripts. Cloning of the synthetic oligonucleotids into the pSuperGFP vector (pS-GFP; OligoEngine) was performed by digesting the empty vector with EcoR1 and Xho1 according to the manufacturer's instruction. Cells were transfected at low density (<20.000 cells/cm²) and expression analysis was performed according to the protocol described by Savaskan et al. [6] (link).

Reverse transcription-polymerase chain reaction was used for full length cloning of PRG3 from rat, mouse and human mRNA samples as described previously [13 (link)]. For sequence alignments and homology searches of PRGs we utilized the www.ncbi.nlm.nih.gov database and A Plasmid editor software (ApE; MW Davis, Utah, USA). Transmembrane domains have been predicted using the Kyte Doolittle algorithm and all orthologous sequences of PRG3 (human, mouse and rat) are deposited at the NCBI database (Human PRG3 GenBank accession no. AY304516; Rattus norvegicus PRG3 GenBank accession no. AY299399; Mus musculus PRG3 GenBank Accession no. AY345342). For construct cloning we cloned fragments by PCR and inserted the resulting amplicons into the pEGFP (Takara, Heidelberg, Germany), and pmRFP (Kes Jalink, NKI, Amsterdam, the Netherlands) vectors. C-terminal domain of PRG3 was cloned by PCR amplification out of the full length clone and inserted the amplicon into linearized pEGFP vectors. According to the criteria of Naito et al. [40 (link)] three 19-mer short interfering RNAs were chosen for RNA interference with rodent PRG3 transcripts (GenBank acc. AY299399). Cloning of the synthetic oligonucleotids into the pSuperGFP vector (pS-GFPneo; OligoEngine, Seattle, USA) was performed by digesting the empty vector with Bgl2 and EcoR1 according to the manufacturer's instruction.

The cDNA of full-length mouse MTCL1 (AK147205) was purchased from Danaform (Kanagawa, Japan). cDNA fragments corresponding to wild-type MTCL1 or its mutants were subcloned into appropriate expression vectors: pCAGGS-V5, pCAGGS-Flag- streptavidin-binding protein (SBP) [24 (link)], pEGFP (Takara Bio Inc.), pTagRFP (Evrogen JSC), pGEX (GE Healthcare), or pSRHA [25 (link)]. To establish point mutations in the N1 fragment (5LP, 5LA, and 5EA), NruI-KpnI cDNA fragments corresponding to the N-terminal region of mMTCL1 were synthesized with the appropriate mutations (Thermo Fisher Scientific) and used to replace the wild-type fragment in the V5-N1 or SBP-N1 expression vector. The amino acid numbers of mouse MTCL1 covered by the mutants are summarized in S1 Table. The C6 mutant of human MTCL1 used in previous studies [21 (link), 22 (link)] was renamed as hCMTBD for convenience.

Human and rodent expression constructs were cloned as described previously in Savaskan et al., 2008 [8 ]). For sequence alignments and homology searches of xCT we utilized the www.ncbi.nlm.nih.gov database and A Plasmid editor software (ApE; MW Davis, Utah, USA). All orthologous sequences of xCT (human, mouse and rat) are deposited at the NCBI database (Human xCT GenBank accession no. AF252872; Rattus norvegicus xCT GenBank accession no. NM001107673; Mus musculus xCT GenBank Accession no. AB022345). For construct cloning we cloned fragments by PCR and inserted the resulting amplicons into the pEGFP (Takara, Heidelberg, Germany) vector. According to the critera of Ui-Tei et al., 2004 [29 (link)] three 19-mer short interfering RNAs were chosen for RNA interference with rat xCT transcripts (GenBank acc. NM001107673). Cloning of the synthetic oligonucleotids into the pSuperGFP vector (pS-GFP; OligoEngine) was performed by digesting the empty vector with EcoR1 and Xho1 according to the manufacturer's instruction. Cells were transfected at low density (<20.000 cells/cm²) and expression analysis was performed as Savaskan et al., 2008 [8 ] described.