Prl sv40 vector

To test the transcriptional activity of HNF1α on the HNF1A-AS1 promoter, an HNF1A-AS1 promoter fragment containing the HNF1α response element (RE) was amplified by PCR from genomic DNA and cloned into the pGL3-Promoter vector (E1761, Promega). The HNF1α-RE was mutated using the Hieff MutSite-Directed Mutagenesis Kit (Yeasen Biotechnology, Shanghai, China). Huh-7 cells pre-infected with Lenti-HNF1α for 24 h were co-transfected with HNF1α-RE-LUC vectors together with the control pRL-SV40 vector (E2261, Promega). Luciferase activity was measured using the Dual-Glo Luciferase Assay System (E2920, Promega) 48 h post-transfection. All constructs were verified by DNA sequencing. The primer sequences for the constructs are listed in Additional file 1: Table S1. At least three independent transfection experiments were carried out for each condition.

The DNA binding capacity of p53 was assayed using the TransAM p53 transcription factor assay kit (Active Motif, Carlsbad, CA), according to the manufacturer's protocol. This enzyme-linked immunosorbent assay (ELISA) assay uses immobilized oligonucleotides containing p53 consensus binding site to detect active p53. A total of 5 μg of each nuclear protein extract was used for this experiment. Additionally, p53 transcriptional activation was assessed based on luciferase reporter constructs harboring the p21/WAF1 (WWP-Luc; #16451) or thePUMA (PUMA Frag1-Luc; #16591) promoter (Addgene, Cambridge, MA), both comprising p53 responsive elements. The empty pBV-Luc vector was used as negative control (plasmid #16539; Addgene). Renilla luciferase activity was measured for transfection efficiency normalization by co-transfecting cells with the pRL-SV40 vector (Promega). HCT116 cells were seeded at 1 × 10⁵ cells/well on 96-well plates, and co-transfected with 100 ng of luciferase reporter constructs and 10 ng of pRL-SV40 vector, using Lipofectamine 3000 (Invitrogen). Cells were treated 24 h after transfection with 8 μM 5-FU and/or 4 μM XMD8-92. Reporter assays were performed 24 to 48 h post-treatment using the Dual-Luciferase Reporter Assay System (Promega).

For the transient assays, 1.0 x 10⁵ cells were transfected using Lipofectamine LTX 2000 (Invitrogen) with 1 μg of each Luciferase construct and 100 ng of pRL-SV40 vector (Promega), according to the manufacturer’s instructions. Firefly and Renilla Luciferase activities were measured in cell lysates 48 hours after transfection using the DualGlo Luciferase Assay System (Promega) on a Veritas TM Microplate Luminometer (Perkin-Elmer) following the manufacturer’s protocol and as shown in our companion paper and as described previously (48 (link), 55 (link)). All experiments were done in triplicate. Ratios of Renilla luciferase readings to firefly luciferase readings were taken for each experiment, and triplicates were averaged. The average values of the tested constructs were normalized to the activity of the empty pGL3-basic vector, which was arbitrarily set at value 1.

Luciferase reporter genes activities responded to Nrf2, NF-κB and AP-1 were detected with Dual-Glo Luciferase Assay System (Promega, Mullion, WI, USA). HEK293T-17 cells seeded in 12-well plate were cotransfected with pGL4.37[luc2P/ARE/Hygro] (Promega)/pAP-1-Luc/pNF-κB-Luc (provided by Professor Jian-ping Ye at Pennington Biomedical Research Center, Louisiana State University, LA, USA) expressing firefly luciferase and pRL-SV40 vector (Promega) expressing renilla luciferase in a 10:1 mass ratio. After the infection of IAV and treatment with DMO-CAP for 24 h, Lysis cells and collect supernatants at 4 °C, 12,000 rpm. Then 10 μl sample supernatant and 40 μl of luciferase reagent was added to white 96-well plate and the firefly luminescence was measured after 10 min on Enspire. Then, 40 μl of Stop & Glo Reagent was added and renilla luminescence was measured in the same plate after 10 min. Luciferase activities were calculated by the ratio of the firefly luminescence to the renilla luminescence.

We investigated whether the rs1279736C>A and rs3756585T>G (−283 and −123 from transcription start site) modulate the promoter activity of receptor for activated C kinase 1 [RACK1] by luciferase assay. A 401bp fragment (from −378 to +21) including rs1279736C>A and rs3756585T>G was synthesized by PCR using genomic DNA from a donor carrying heterozygote at both SNPs. The forward primer with KpnI restriction site (5′-GGGGTACCAATTAAGCTCCCCTGGGGTTG-3′) and the reverse primer with HindIII restriction site (5′-CCCAAGCTTCCGCCTTGCAGTGAAAGAGA-3′) were used. The PCR products were cloned into the KpnI/HindIII site of the pGL3-basic plasmid (Promega, Madison, WI, USA). The correct sequence of all the clones was verified by DNA sequencing. The NLSCL cell line, H1299, was transfected with pRL-SV40 vector (Promega, Madison, WI, USA) and pGL3-basic plasmid using Effectene transfection reagent (Qiagen, Hilden, Germany). The cells were collected 48 h after transfection, and the cell lysates were prepared according to Promega's instruction manual. The luciferase activity was measured using a Lumat LB953 luminometer (EG & G Berthhold, Bad Wildbad, Germany), and the results were normalized using Renilla luciferase activity. All experiments were performed in triplicate.

For the transient assays, 1.0 x 10⁵ cells from both cell lines were transfected using Lipofectamine LTX 2000 (Invitrogen) with 1 μg of each Luciferase construct and 100 ng of pRL-SV40 vector (Promega), according to the manufacturer’s instructions. Firefly and Renilla Luciferase activities were measured in cell lysates 48 hours after transfection using the DualGlo Luciferase Assay System (Promega) on a Veritas TM Microplate Luminometer (Perkin Elmer) following the manufacturer’s protocol. All experiments were done in triplicate. Ratios of Renilla luciferase readings to firefly luciferase readings were taken for each experiment, and triplicates were averaged. The average values of the tested constructs were normalized to the activity of the empty pGL3-basic vector, which was arbitrarily set at value 1.