Plenti 4.1 ex mir200c 141

MDA-231EV cells were created by infecting MDA-MB-231 cells with copGFP lentiviral particles (cat sc-108,084, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and stable cells were selected by continual culture in 5 µg/mL puromycin (InvivoGen, San Diego, CA, USA). MDA-231c141 cells were created by transfecting MDA-MB-231 cells with pLenti 4.1 Ex miR200c-141 (cat #35,534, Addgene, Watertown, MA, USA) and stable cells were selected by continual culture in 5 µg/mL puromycin (InvivoGen, San Diego, CA, USA). The in vitro characterization of MDA-231EV and MDA-231c141 cells have previously been described [29 (link)].

pLenti 4.1 Ex miR200c-141 was purchased from Addgene (#35534. Cambridge, MA, USA). The human miR141 cDNA was cloned and inserted into pLenti 4.1 between the EcoR I and Xho I sites to generate pLenti 4.1-miR141 plasmid. The 3′-UTR of human EGFR cDNA (hEGFR-3′-UTR) was cloned and inserted into pIS0 vector [37 (link)] (#12178. Addgene. Cambridge, MA, USA) between the Sac I and Xba I sites to generate pIS0-hEGFR3′UTR plasmid. The promoter of human EGFR gene (hEGFRp) was cloned and inserted into PGL3b vector between the Kpn I and Nar I sites to generate pGL3b-hEGFRp plasmid. The miR141 sponge was inserted into the pBABE- puro-mCherry vector between the EcoR I and Age I sites to generate pBABE-puro-mcherry-miR141sponge plasmid. The human miR141 promoter (hmiR141p) was cloned and inserted into pGL3b between the Nhe I and Hind III sites to generate pGL3b-hmiR141p plasmid.