Pcmv6 entry vector

For BMP-2 overexpression, Caki-1 and Caki-2 cells were transfected with a pCMV6-ENTRY vector expressing human BMP-2 cDNA or an empty pCMV6-ENTRY vector (OriGene Technologies, Rockville, MD) using X-treme gene HD Transfection Reagent (Roche Diagnostics, Indianapolis, IN), according to the manufacturer's protocol.

DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human PMS2 cDNA as well as empty pCMV6-ENTRY vector as a control (Origene) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol. Clones were selected using 500 μg/ml of G418 (Invitrogen). Colonies resistant to G418 appeared within 2 weeks and single colonies were picked and then expanded for another 3 weeks to make stable clone stock cells.

Enhanced chemiluminescence (ECL) reagents, nitrocellulose membranes (Hybond-C extra), and secondary Cy3-conjugated antibodies were from Amersham Pharmacia Biotech Inc. (Piscataway, NJ, USA). Monoclonal antibody against SERPINB3 (sc-21767), LaminA (sc-20680) and PCNA (sc-25280) or polyclonal antibody for HIF-1α (sc-53546) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibody for HIF-2α and HO-1 were from Novus Biologicals (Cambridge, UK) and Stressgen (Victoria, BC, Canada), respectively. Monoclonal antibodies for α-tubulin and β-actin and all other reagents of analytical grade were from Sigma Chemical Co (Sigma Aldrich Spa, Milan, Italy), Lipofectamine 2000 (Invitrogen-Life Technologies), Plasmid DNA purification NucleoBond XtraMIDI (Macherey-Nagel, Germany), pCMV6-Entry vectors (Origene, Rockville, MD), Boyden's chambers were from Neuro Probe, Inc. (MD, USA).

For the overexpression experiments, pCMV6 entry vectors containing the open reading frames (ORF) of the five candidate mouse proteins mOCIAD1, mPAFAH2, mHTATIP2, mPDCD6 and mSAR1B were purchased from Origene Technologies GmbH (Herford, Germany). To equip the proteins with C-terminal myc-tags, the ORFs were amplified by RT-PCR, using gene-specific primers equipped with appropriate restriction cites (see Table S3), and recloned into pCMV3A mammalian expression vectors (Agilent Technologies, Santa Clara, CA, USA). The correct insertion and preservation of the ORF was subsequently checked by sequencing (Eurofins Genomics, Ebersberg, Germany).
For the colocalization experiments, HepG2 cells and mouse embryonic fibroblast (for preparation see [18 (link)]) were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 1% penicillin/streptomycin. For immunofluorescence microscopy, the cells were seeded into 24-well cell culture plates (Sarstedt, Nümbrecht, Germany) equipped with Poly-D-lysine-coated (Thermo Fisher Scientific, Waltham, MA, USA) glass coverslips. For overexpression experiments, the cells were transfected on the next day with LipofectamineTM3000 reagent (Thermo Fisher) containing 0.5 μg plasmid DNA per well. After 24 h of incubation, the cells were fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 20 min, at RT.