Plasmids, shRNA, Lentivirus Production, and Transduction

For the over-expression of CENP-U, the coding sequence of human CENP-U (Biolight, Nanjing, China) was cloned into the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro (System Biosciences, CA, USA). According to the manufacturer’ instructions, a mixture of CMV-dR8.91, pCMV-VSV-G, and pCDH-CENP-U or pCDH-CMV-MCS-EF1-Puro was transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen) to produce lentivirus. T47D and MCF-10A cells were infected with recombinant lentivirus-transducing plus 8 mg/ml polybrene (Sigma). For the knockdown of CENP-U, shRNA plasmid, shControl plasmid, and lentiviral packaging system were bought from Genechem (Shanghai, China). According to the Genechem’s manufacturer’s instructions, the packaged lentiviruses were obtained after shCENP-U/shControl cotransfection with lenti-Easy Packaging Mix for 48 h to infect MDA-MB-231 and MCF-7 cells.