Pebmulti neo vector

pEB Multi-Neo vector (Wako), in which genes encoding PDK2 with a C terminus HA tag and IRES-DsRed-express2 (Clontech) were inserted, was transfected into HMECs using Lipofectamine 2000. The pool of transfected cells was passaged once before a double sorting of DsRed-expressing cells via fluorescence activated cell sorting. The sorted cells were passaged once before proceeding to the WST-1 proliferation assay. A pEB Multi-Neo vector with IRES-DsRed-express2 was used as the negative control vector.

The method used to establish cells in which IL-32 is continuously highly expressed is mentioned below. Using the first-strand cDNA derived from MIA PaCa-2 cells with low invasive potential as a template, human IL-32 cDNA was amplified by PCR with primers 5′-ggggtaccGCCATGTGCTTCCCGAAGGTC CTCTCTG-3′ (the KpnI site is underlined) and 5′-gggcggccgc TCATTTTGAGGATTGGGGTTCAGAG-3′ (the NotI site is underlined) and subcloned into pLITMUS 28 plasmid vector (New England Biolabs). After confirmation of the cDNA sequence using an ABI3500 Genetic Analyzer (Thermo Fischer Scientific, Inc.), the KpnI- and NotI-digested IL-32β and IL-32ε cDNA fragments were cloned between the KpnI and NotI sites of the pEBMulti-Neo vector (FUJIFILM Wako Pure Chemical Corporation), to yield the plasmids pEB-IL-32β and pEB-IL-32ε. Then, the pEB-IL32β, pEB-IL32ε and control pEB Multi-Neo plasmids were transfected into P cells with Lipofectamine 3000 reagent (Thermo Fischer Scientific, Inc.), according to the manufacturer's protocol. After 48 h, the transfected P cells were selected with G418 (500 µg/ml) for 2 weeks, yielding IL-32β-overexpressing (β), IL-32ε-overexpressing (ε) and control cells with the unmodified plasmid (MOCK).