Pcmv6 e2f1

The human CDC20B promoter was cloned into the pGL3 Firefly Luciferase reporter vector (Promega) with SacI and NheI cloning sites. The promoter sequenced ranged from −1073 to +104 relative to the transcription start site. 37.5 ng of pGL3 plasmid were applied per well. pCMV6-Neg, pCMV6-E2F1 (NM_005225) and pCMV6-E2F4 (NM_001950) constructs were from Origene. 37.5 ng of each plasmid was applied per well. 25 ng per well of pRL-CMV (Promega) was applied in the transfection mix for transfection normalization (Renilla luciferase). HEK 293T cells were seeded at 20,000 cells per well on 96-well plates. The following day, cells were transfected with the indicated plasmids (100 ng of total DNA) with lipofectamine 3000 (Invitrogen). After 24 h, cells were processed with the DualGlo kit (Promega) and luciferase activity was recorded on a plate reader.

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.