Pgl4.21 vector

Human TRIM72 promoter (chr16: 31224983–31225673, hg19) and rhesus TRIM72 promoter (chr20: 28866360–28867045, rheMac8) were cloned using genomic DNA extracted from human VSMC cells and rhesus heart tissues.
For the in vitro luciferase reporter assay, original TRIM72 promoters, promoters with truncations (supplementary table S3, Supplementary Material online) or point mutations (supplementary tables S4 and S5, Supplementary Material online) were cloned into pGL4.21 vector (E676A, Promega), and the SV40 enhancer was replaced with a TRIM72 downstream element (chr16: 31226063–31226476, hg19). Mutation vectors were constructed with mutated primers using Gibson assembly (C215, Vazyme).
For the in vivo LacZ reporter assay, human and rhesus TRIM72 promoters were cloned into a modified pWHERE vector (He et al. 2011 (link)) (H19-Hsp68 miniP-LacZ-ΔCpG NLS-H19, InvivoGen) using HindIII and XhoI. All constructs were Sanger sequenced using plasmid DNA isolated from transformed bacterial cells.

Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal calf serum (FCS) were purchased from PAN-Biotech (Aidenbach, Germany). Phosphate-buffered saline (PBS) and medium supplements (glutamine, non-essential amino acids, penicillin/streptomycin) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Rifampicin, crystal violet, and dimethylsulfoxide (DMSO) were purchased from Applichem (Darmstadt, Germany) and methanol from Roth (Karlsruhe, Germany). Dovitinib was provided by Sequoia Research Products (Pangbourne, UK). Triptolide was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Oxaliplatin (dissolved in distilled water) was supplied by the University Hospital’s pharmacy. The Dual-Glo Luciferase Assay System, the pGL4.21 vector, the pGL4.74 [hRluc/TK] Renilla vector, and the FuGene® HD Transfection reagent were purchased from Promega Corporation (Madison, WI, USA). The NR1I2 (NM_003889) human cDNA TrueClone® (pCMV6-XL4 vector, containing the cDNA of the PXR gene NR1I2) was obtained from OriGene (Rockville, MD, USA). Cell culture flasks and white 96-well plates with a white bottom (especially well-suited for luminescence measurements) were obtained from Greiner (Frickenhausen, Germany). The luminescence signal was detected with the SpectraMax iD3 from Molecular Devices (Wokingham, UK).

The construction and basic principle of the PXR reporter gene vector have been described and published previously (Weiss et al. 2013 (link)). Briefly, the proximal response element module (PREM, comprising −362/+53 region) and the xenobiotic response element module (XREM, comprising −7836/−7208 region) of the CYP3A4 promoter had been cloned upstream of the firefly open reading frame of the pGL4.21 vector (Promega, Mannheim, Germany). The pGL4.74 [hRluc/TK] Renilla vector was used as a normalization vector to control for transfection efficiency. To ensure high expression of PXR, cells were co-transfected with pCMV6-XL4 containing the cDNA of human PXR (NR1I2).
For transfection, 50,000 cells per well were seeded and allowed to attach and grow overnight. The next day, the medium was replaced by a medium without supplements. Four hours later, transfection was performed with the lipid-based transfection reagent FuGene®. The ratio of transfection reagent to DNA was 5:1. Each well was exposed to 20 ng of the PXR expression vector, 80 ng of the reporter vector, and 10 ng of the Renilla vector. After the addition of the transfection reagent-DNA mix, the plate was shaken for 30 s at room temperature. Cells were then incubated at 37 °C for 24 h.