Pcdh cmv mcs ef1 cogfp construct

Human CRC cell lines, including SW480, SW620 and LOVO cells were purchased from the American Type Culture Collection (Manassas, VA, USA). HCT-116, LS-174T and HT29 were purchased from the Shanghai Cell Biology, University of the Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) that is supplemented with 10% fetal bovine serum (Gibco, CA, USA) in a humid wet atmosphere containing 5% CO₂ at 37°C. ANNEXIN V-FITC/7-AAD KIT was purchased from Beckman Coulter. A full-length PRRX1 cDNA that entirely lacked the 3′-UTR was purchased from GeneCopeia (Rockville, MD, USA) and subcloned into the eukaryotic expression vector pcDNA3.1 (+) (Invitrogen, USA). The pre-miR-124 sequence was amplified and cloned into pCDH-CMV-MCS-EF1-coGFP constructs (System Biosciences, California, USA). The virus particles were harvested 48 h after transfecting pCDH-CMV-miR-124 with the packaging plasmid pRSV/pREV, pCMV/pVSVG, and pMDLG/pRRE into 293T cells using Lipofectamine 2000 reagent (Invitrogen, USA). PRRX1 knockdown and control lentiviruses were purchased from GENECHEM (Shanghai, China). MiR-124 mimic, a non-specific miR control, anti-miR-124, and a non-specific anti-miR control were purchased from Thermo Scientific Dharmacon(USA).