Pcmv6 c myc

pCMV6-E2F1, pCMV6-c-myc, and pCMV6-XL5 empty plasmid was purchased from Origene (Rockville, MD). MiR-301a hairpin antagomirs were obtained from GeneCopoeia Inc. (Guangzhou, China). All transfections were conducted using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). miR-301a-expressing retroviruses, Cip2a-expressing retroviruses were purchased from GENECHEM (Shanghai, China). The Cip2a short hairpin RNA (shRNA) lentivirus vectors were generated by GeneCopoeia Inc. (Guangzhou, China). The target sequences of Cip2a shRNA was 5'-CUGUGGUUGUGUUUGCACUTT-3'. For selection of TNBC stable cell lines, retroviruses were transduced into BT549 and MDA-MB-231 cells in the presence of polybrene (6 μg/mL, Sigma). Cells were selected with 1 μg/mL puromycin for 7 days.

Lentiviral vectors expressing nontargeting pLKO.1 control shRNA (SCH002), and two shRNA constructs targeting NNMT-shRNA1 (TRCN0000035224) and -shRNA2 (TRCN0000035225) were obtained from Sigma-Aldrich. Expression plasmid for pCMV6-NNMT, pCMV6-SIRT1, pCMV6-c-myc, and pCMV6-XL5 empty plasmid were purchased from Origene (Rockville, MD). For cell transfection, Lipofectamine 2000 reagent (Invitrogen) was used according to the manufacturer’s instructions. After 72 h infection, stably expressing or knockdown cells were selected in RPMI 1640 medium containing 1 μg/mL puromycin.