Pcmv6 ac gfp vector

A549, H460 and H1299 cells were purchased from ATCC and cultured in RPMI-1640 medium supplemented with 10% FBS and penicillin/streptomycin. For stable clones generation, cells were transfected using SureSilencing shRNA plasmids (Qiagen, Valencia, CA, USA) and cells stably integrating the silencing plasmid were selected and sub-cultured in medium containing 1 mg/ml of puromycin. HeLa-DR13-9 cells were cultured as previously described [50 (link)]. Plasmid and siRNA transfections were performed using Lipofectamine-2000 or Oligofectamine (Invitrogen, Carlsbad, CA, USA), respectively, according to manufacturer's instructions. Control and anti-RanBP9 siRNAs were purchased from Santa-Cruz (Santa Cruz, CA, USA). Plasmid for transient expression of RanBP9-GFP fusion protein, cloned in pCMV6-AC-GFP vector, was purchased from Origene. For irradiation experiments, cells were treated using an X-ray linear accelerator (Gammacell 40 Exactor, Best Theratonics, Ottawa, Canada) with doses ranging from 1 to 10 Gy. For ATM inhibition, cells were treated with 10 μM KU-55933 (EMD-Millipore, Billerica, MA, USA) for 1 h before ATM activation.

The murine cancer cell lines for melanoma (B16F10, referred to as B16), and colon cancer (CT26) were obtained from ATCC. Cells were maintained in RPMI medium supplemented with 10% fetal calf serum (FCS) and penicillin with streptomycin (complete RPMI media). B16^IDO and B16^TDO cells were generated by transduction of B16F10 with GFP plus the IDO gene or TDO gene in the pCMV6-AC-GFP vector (Origene). B16F10 transduced with GFP alone were used as control cells (B16-WT). Clonal transformants were selected using 1 mg/ml G418 (Geneticin). Cell lines were routinely screened to avoid mycoplasma contamination and maintained in a humidified chamber with 5% CO₂ at 37 °C for up to 1 week after thawing before injection in mice.