Pllu2g

PAa lung adenocarcinoma cells were obtained from Peking University Health Science Center (Beijing, China), and the 293FT human embryonic kidney cell line was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Lentiviral vectors [pLLU2G-green fluorescent protein (GFP)], packaging systems (3rd generation lentivirus packing system) and negative control virus particles (pLP1, pLP2, pLP/VSV-G and pLLU2G) were obtained from Invitrogen (Thermo Fisher Scientific, Inc.) Lipofectamine^® 2000 transfection reagent and One Shot^® Stbl3™ chemically competent E. coli were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The QIAquick Gel Extraction Kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and diethylpyrocarbonate were all purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany).

For the silencing of CyPA expression, DNA oligonucleotides were designed based on the CyPA siRNA sequence (5′-TCTCGAGTTTTTCTCGAGA-3′), and cloned into pLLU2G lentiviral vectors to construct pLLU2G-CyPA small hairpin (sh)RNA plasmids, according to the method reported previously (15 (link)). Briefly, DNA oligonucleotides were ligated with plasmid pLLU2G and digested with HpaI and XhoI to form double stranded DNA. The double stranded DNA was then transformed into One Shot Stbl3™ Chemically Competent E. coli (Thermo Fisher Scientific, Inc.). Negative control virus particles (pLP1, pLP2, pLP/VSV-G and pLLU2G) from Invitrogen (Thermo Fisher Scientific, Inc.) were used to monitor the nonspecific reactions induced by the shRNA, and to optimize the efficiency of virus transduction according to the manufacturer's protocol.