Human lung cancer H5800 cells were obtained from Dr Luo (Boston University School of Medicine, Boston, MA, USA), who originally purchased it from ATCC ((Manassas, VA, USA). LKR murine lung cells are a generous gift from Dr Jacks (MIT, Cambridge, MA, USA) and were also used by other groups (Johnson et al, 2001 (link); Matsumura et al, 2010 (link); Nishioka et al, 2010 (link)). The lung cancer cells were cultured in DMEM medium containing 10% fetal calf serum. Nicotine, cisplatin and inhibitors were purchased from Sigma (St Louis, MO, USA). Nicotine was dissolved in 1 × PBX and the concentration of 0.5 μM was used in previous studies (Nishioka et al, 2010 (link), 2011 (link)).
Short hairpin (sh) RNA for bcl-2 was introduced into a lentiviral vector (OriGene, Rockville, MD, USA) that contains the 19 bp target sequence for bcl-2 (5′-GTGGATGACTGAGTACCTG-3′) (Wild-Bode et al, 2001 (link); Kock et al, 2007 (link)), or scrambled sequence. The lentiviral construct was transfected into 293T cells. Forty-eight hours later, the supernatant was collected and tittered by serial dilutions. The supernatant containing optimal concentration of the viral particles was used for the knockdown experiments. For Bcl-2 overexpression, wt-bcl-2 was constructed in the PCDNA vector and subsequently transfected into the cells.
Short hairpin (sh) RNA for bcl-2 was introduced into a lentiviral vector (OriGene, Rockville, MD, USA) that contains the 19 bp target sequence for bcl-2 (5′-GTGGATGACTGAGTACCTG-3′) (Wild-Bode et al, 2001 (link); Kock et al, 2007 (link)), or scrambled sequence. The lentiviral construct was transfected into 293T cells. Forty-eight hours later, the supernatant was collected and tittered by serial dilutions. The supernatant containing optimal concentration of the viral particles was used for the knockdown experiments. For Bcl-2 overexpression, wt-bcl-2 was constructed in the PCDNA vector and subsequently transfected into the cells.