A DUSP16 expression plasmid was made by subcloning human DUSP16 cDNA to a pPyCAGIP vector. The pPyCAGIP vector confers resistance to puromycin via a puromycin resistance gene to allow for selection of positively transfected clones. Oligonucleotide primers (Integrated DNA Technologies) for DUSP16 with XhoI and NotI restriction sites were used to amplify the full-length cDNA of the DUSP16 gene. The forward primer was synthesized with an XhoI restriction site and the reverse primer with a NotI restriction site (Supplementary Table 2).

Cancer cell lines were transfected with either empty pPyCAGIP vector or with the DUSP16-pPyCAGIP vector using Lipofectamine® LTX (Invitrogen). Transfected cells were selected using media containing puromycin. Selected single cells were isolated and assessed for DUSP16 expression by quantitative real-time PCR (qPCR) and western blot.

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