Pgl4 basic vector

For construction of caspase 8 promoter fragment (0.5 kB) luciferase-reporter plasmids we amplified using genomic DNA from jurkat cells as template by PCR approach and oligonucleotides that are flanked by KpnI and HindIII restriction sites. The resulting fragments were then cloned into the KpnI and HindIII sites of the pGL4-Basic vector (Promega). Promoter sequences amplified by PCR were confirmed by DNA sequencing using Mayo sequencing core lab. The TK-Renilla expression vector purchased from Promega (Madison, WI), was used as an internal control for transfections. Primary CD4 cells were transfected with 10 ug plasmid (9.5 mcg Casp8-luc and 0.5 mcg TK-renilla luc/10⁶ cells using an Electro Square Porator T820 (BTX, San Diego, CA) electroporator. Cells were then primed with Flurbiprofen, Indomethacin, Doxycycline, Bezafibrate, DMSO or PHA (positive control). Results are expressed as ratio of casp8-luc/Renilla expression at 24hours[50 (link)].

The methods used for the construction of the CXCL10 gene promoter-driving luciferase reporter, wild-type pCXCL10-Luc(−250/+8)) and disruption of the NF-κB-binding site by site-directed mutagenesis, pCXCR3-Luc(−178/+22)mtNF-κB, are described elsewhere [26 (link)].
For the generation of CXCR3 gene promoter-driving luciferase reporter constructs, the CXCR3 promoter fragment spanning nucleotides −178 to +22 upstream of the transcription start site was synthesized from human genomic DNA (Promega, Madison, WI, USA) via PCR using the primers 5′-ggtaccCATCCTCTGCCAGCTTTTCT-3′ (forward primer) and 5′-agatctCTTTGGTGCTTGTGGTTGGA-3′ (reverse primer). Small letters indicate inserted KpnI and BglII restriction sites. The PCR products ligated into a T&A vector (RBC Bioscience, Taipei county, Taiwan) were digested by KpnI and BglII and cloned into the KpnI and BglII sites of the pGL4-basic vector (Promega), yielding pCXCR3-Luc(−178/+22). Site-specific mutation of two NF-κB binding sites, mtNF-κB(I) and mtNF-κB(II), was performed using an EZchange Site-directed Mutagenesis Kit (Enzynomics, Daejeon, Republic of Korea), using the −178/+22 construct as a template plasmid. Primer sequences used to generate point mutations were as Table 2.
RT-PCR was carried out by annealing at 55 °C for 25 cycles. The point mutation was verified by DNA sequencing (Macrogen, Seoul, Korea).

FLAG-tagged HMMR expression vector was constructed by inserting PCR amplified HMMR fragment into the pcDNA3 vector (Invitrogen) linked with FLAG tag at the amino terminus. The HMMR promoter luciferase reporters were made by inserting PCR-amplified HMMR promoter fragments into the pGL4-Basic vector (Promega). HepG2 and MHCC-97H liver cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Small interfering RNAs (siRNAs) were synthesized by JTS scientific or GemmaPharma. The cDNA target sequences of siRNAs and/or short hairpin RNAs (shRNAs) for HMMR and CEBPα were listed in Table S1. Stable cell lines overexpressing HMMR shRNA were established by lentiviral transduction using pSIH-H1-Puro carrying HMMR shRNA. Anti-cyclin D1, anti-cyclin E, anti-cyclin B1 and anti-CEBPα were purchased from Santa Cruz Biotechnology; Anti-HMMR was from Proteintech.