Ter lysis buffer into each well, the plate was placed at -80 for 30 min and then equilibrated at room temperature. The cellular lysate was centrifuged at maximum speed for 1 min. Cleared lysates were used to measure luciferase and renilla activity on GloMax?20/20 Luminometer (Promega, E5331) by DualLuciferase Reporter Assay System (Promega, E1910).Overexpression of Dll4-ASNE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific #78833) was used to separate nuclei and cytoplasm of MS1 cells. The separated two parts were subjected to TRIzol extraction, followed by reverse transcription. Xist served as positive control for nuclear compartment and tRNA-Met for cytoplasmic compartment. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26100631 The primers were Xist-for, tgcgggttcttggtcgatgt, Xist-rev, cgcttg agatcagtgctggc; tRNA-Met-for, ggcccataccccgaaaac, tRNAMet-rev, acgggaaggatttaaaccaa.Quantitative PCRpTracerTM-CMV2 Vector (Life technologies, V885-01) was digested with EcoRV and NotI for sub-cloning Dll4-AS isoforms. Dll4-AS1, -AS2 and -AS3 were amplified from MS1 cDNA using Phusion DNA polymerase and the primers harbored NotI site at the 3 end. The plasmid contained a separate cassette encoding green fluorescent protein (GFP) -Zeocin expression that allowed for stable selection of transfected cells. Transfection of the plasmids was performed according to the protocol of Lipofecatmine 2000 (Life technologies, 11668027). The cells were Caspase-3 Inhibitor cost selected by Zeocin, and cells overexpressing Dll4-AS were tracked through GFP expression.Dll4-AS knockdownTotal RNA from cultivated cells was extracted by TRIzol reagent followed by DNase I treatment for 2 h at 37 . The DNA-free RNA was further purified using RNAeasy Mini kit (Qiagen, 74104). RNA concentrations were measured by Nanodrop (Thermo Scientific), followed by reverse transcription by SuperScript III (Life Sciences). Quantitative PCR was carried out with SYBR Green I in iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, 170?780). The expression levels were normalized to internal actin or CD31. Primers for mouse actin, Dll4-AS1, -AS2, -AS3, total Dll4-AS and Dll4 were: actin-for, ctcttttccagccttccttct, actin-rev, aggtctttacgga tgtcaacg; AS1-for, ttctcaaaaactccgctgct, AS1-rev,ctctgctct ttcccctcctc; AS2-for, atccgacgccttaacctttc, AS2-rev,ctccgt tctgctcctattgc; AS3-for, cccgaaaccttgacttttca, AS3-rev, ccac cagaggataggagggta; ASt-for, gaggcaataggagcagaacg, ASt-rev,Vector-Based miRNA and synthetic siRNA were used to knockdown Dll4-AS expression in both MS1 and EOMA cells. pcDNA6.2-GW/EmGFP-miR (Life technologies, K4936-00) was used to express miRNA targeting the common last exon of all Dll4-AS isoforms. The inside single-stranded DNA oligonucleotides encoding the target pre-miRNA and the complementary oligonucleotides for miR1 are tgctgaggttgttcagtcaagaacctgttttggccactgactgac aggttcttctgaacaacct and cctgaggttgttcagaagaacctgtcagtcagt ggccaaaacaggttcttgactgaacaacctc, respectively. Those for miR2 are tgctgctctgattagatccattcaaggttttggccactgactgaccttg aatgtctaatcagag and cctgctctgattagacattcaaggtcagtcagtggcc aaaaccttgaatggatctaatcagagc, respectively. The forward and reverse synthetic siRNAs for control, siRNA1 and siRNA2 are ggugagccguguagaguaatt, uuacucuacacggcucacctt,Li et al. Vascular Cell (2015) 7:Page 3 ofgguaggaggccugugauaatt, uuaucacaggccuccuacctt, ggaggccug ugauaagguutt, aaccuuaucacaggccucctt, respectively.ImmunostainingCell migration assayMS1 cells were plated on sterile glass coverslips placed in the wells of a culture plate and allowed to adh.