Pgl3 promoter plasmid

An 3xSTAT3/Luc fragment containing tandem repeats of double-stranded oligonucleotides spanning the STAT3 binding site (5′-CATTTCCCGTAAATC-3′) [30] (link) was amplified with the primers sense: 5′-CATT TCCCGTAAAT CCATTTCCCGTAAATCCATTTCCCGTAAATC-3′ and then introduced into the pGL3 promoter plasmid (Promega, Madison, WI). All transfection experiments were performed using Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's instructions. For luciferase assays, the cell lysate was first mixed with luciferase substrate solution (Promega), and the resulting activity was measured using a luminometer. For each experiment, luciferase activity was determined in triplicate and normalized using β-galactosidase activity.

The MET 3′-UTR reporter plasmid was a kind gift from Dr. Lin He (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) [16] (link). The EGFR 3′-UTR reporter plasmid was a kind gift from Dr. Benjamin Purow (University of Virginia, VA) [17] (link). It was constructed via insertion of the EGFR 3′-UTR into the XbaI restriction site 3′ to luciferase in the pGL3-promoter plasmid (Promega Corp.). The Bcl-xL 3′UTR-Luc plasmid was purchased from GeneCopoeia (Rockville, MD).

Plasmid end-joining assay was conducted as previously described^{63 (link)}. The pGL3-promoter plasmid (Promega), which harbors a luciferase reporter gene, was linearized by HindIII and confirmed by agarose gel electrophoresis. The linearized DNA was purified by gel extraction kit (Qiagen), dissolved in sterilized water, and transfected into cells after knockdown of target genes for three days. Luciferase protein was expressed when the cutting sites were repaired by end-joining. The luciferase activity was assayed by Luciferase Assay System (Promega). A Renilla plasmid was co-transfected as a control.

The Gal3 promoter (NM_001357678), Notch1 promoter (NM_017617.5), and Jag1 promoter (NM_000214) were amplified by PCR with HUVECs DNA as a template, and subcloned into the pGL3-basic plasmid (Promega, Madison, WI, USA) to construct pGL-Gal3 and pGL-Jag1. Amplified fragments were cloned into the pGL3-promoter plasmid (Promega, Madison, Wisconsin, USA) to construct a correspondent plasmid. HEK293T cells were co-transfected with pGL-Gal3 and pGL-Jag (100 ng).

The oligonucleotides containing short hairpin RNA (shRNA)
sequences, targeted against rat Cmtr1, Cpeb2, or a nontarget control, were constructed into pLentiLox (LL)3.7/Syn plasmid with HpaI and XhoI restriction
enzymes cutting sites for cloning. The (GCAGCCCTGCTCTGATGGT) shRNA
clone against Cmtr1 and (GCAGAAAGCAAGTCCTATT) shRNA
clone against Cpeb2 were designed for gene targeting.
For a reporter assay, mouse PDGFRα 3′-UTR was PCR-amplified
from lung cDNA for further construction. The amplified DNA fragment
was cloned to the pGL3 promoter plasmid (Promega) using XbaI and SalI cloning sites. The resulting plasmid was digested with
XbaI and self-ligated to generate the 3′UTR 1-kb construct.
The plasmid pT7-EGFP-N1 used for in vitro transcription
was constructed by linearization of pEGFP-N1 (Clontech) with NheI
and BgIII followed by ligation with the annealed oligonucleotides
of the T7 promoter (5′-CTAGTAATACGACTCATATAGGGA-3′ and
5′-GATCTCCCTATATGAGTCGTATTA-3′). For pLL3.7/Syn-mCherry and pLL3.7/Syn-Gfp, the cytomegalovirus (CMV) promoter in the
pLL3.7 plasmid was placed with mCherry to produce
pLL3.7-Syn/mCherry. For pGW1-HA-Sbf1, Sbf1 was amplified by specific primers, forward 5′-GCTGGTCGACATGGCGCGGCTCGCGGAC-3′,
and reverse 5′-GGAATTCGGCATCCGACAGGCAGC-3′, with flanking
of SalI and EcoRI restriction enzyme
cutting sites. The plasmids used in this study were listed in Table S5.