Phrl tk

In Table 1, the following four synthetic, chemically modified short RNA oligonucleotides were purchased from Shanghai GenePharma Co. Ltd. The Dual-Luciferase® Reporter Assay System and phRL-TK were obtained from Promega (Beijing) Biotech Co. Ltd. The pMIR-REPORT Luciferase Vector was supplied by Thermo Fisher Scientific Co. Ltd. In addition, all the nucleotides used in this work were synthetized by Sangon Biotech Company. Other chemicals were provided by Harbin Baijiesi Technology Co. Ltd.

The pGL3-MALAT1-P1-Luc construct, the pGL3-MALAT1-P2-Luc construct, and the mut-Luc construct were purchased from Genechem (Shanghai, China). The plasmid phRL-tk (used as internal control for transfection efficiency and cytotoxicity of test chemicals) containing the Renilla luciferase gene was purchased from Promega (Madison, WI, USA). The cells proliferated to 60 – 80% confluence after 24 h of culture. Then, the cells were co-transfected with 2 μg of DNA of the reporter constructs and HIF-2α plasmid or HIF-2α siRNA using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The amounts of luciferase and Renilla luciferase were measured with the Dual-Luciferase Reporter Assay System Kit (Promega) following the manufacturer's instructions. The values of luciferase activity for each lysate were normalized to the Renilla luciferase activity. The relative transcriptional activity was converted into fold induction above the vehicle control value.

Human CYFIP1A promoter was synthetized by Blue Heron Biotech (Bothell, WA) and inserted in pGL3-basic (Promega) between the KpnI and BglII restriction sites (pGL3-CYFIP1A-3kb). SCC13 cells or HKCs were co-transfected with 0.5 μg of pGL3-CYFIP1A-3kb, 2 μg of the Notch1 expressing plasmid pcDNA3-Notch1 or empty vector as control and 0.05 μg of the Renilla internal control plasmid (phRL-TK, Promega). As a control of experiment, cells were also in parallel co transfected with 0.5 μg of the RBPjk-luc plasmid (Notch reporter plasmid), 2 μg of the Notch1 expressing plasmid pcDNA3-Notch1 or empty vector as control (pcDNA3) and 0.05 μg of phRL-TK. Cells were harvested 30 hours after transfection and assayed for Firefly and Renilla luciferase activity with the Dual Luciferase assay reporter kit (Promega). Results are expressed as relative firefly activity over Renilla luciferase activity. All experiments were performed in triplicate.

AtT-20, 293T, HeLa, Neuro-2a and CHO cells, purchased from ATCC, were grown in DMEM supplemented with 10% FBS and maintained in the 37 °C incubator containing 5% CO₂. For CRH induced signal activation Assay, after treatment with hCRH and tsCRH peptides (China peptides, Shanghai, China) for different time courses (0, 5 min, 15 min, 30 min, 1 h) or doses (50 nM, 150 nM, 300 nM), cells were lysed in RIPA Buffer (Sigma, Santa Clara, California) containing phosphatase and protease inhibitor cocktails (Pierce, Thermo, grand island, New York), samples were collected in loading buffer (Bio-Rad, Hercules, California) and heated at 100 °C for 5 min. For transfection, 12 h after seeding, cells were co-transfected with reporter vector and a control plasmid phRL-TK (Promega, Madison, Wisconsin) using X-treme 9 DNA transfection reagent (Roche, Basel, Switzerland) according to the producer’s manual.

One hundred uL of H. zea fatbody cells were seeded onto wells of 96-well plates (5 × 10⁵ cells/well). After 30 min, the seeded cells were transiently co-transfected with either of the 6 wildtype (pGL3-CCE001j promoter, pGL3-CCE001b promoter, pGL3-CCE001f promoter, pGL3-CYP6AE19 promoter, pGL3-UGT40F2 promoter, pGL3-ALDH1A1L promoter) and 4 deletion promoter-pGL3 constructs (del-Motif 1, del-Motif 2, del-ARE1 and del-ARE2) (0.1 µg/well) and the internal renilla luciferase control reporter plasmid PHRL-TK (Promega, 0.01 µg/well). There were three technical replicates in both the experimental group and the control group. Six hours after transfection, the final concentration of 18.5 μM flavone (induced) or equivalent volume of methanol (control) was added. After 48 h, the cells were harvested and the luciferase activity of kidney and firefly was measured using the dual luciferase reporting assay system (Promega, Madison, WI, USA) on a TD-20/20 single-tube luminescence instrument designed by Turner (Turner Biosystems, Sunnyvale, CA, USA). The relative firefly luciferase activity normalized against the renilla luciferase activity was calculated as an indicator of the basal or flavone-inducible promoter activity of each construct. At least 3 repeated measurements were conducted for each independent transfection.

HEK293 cells (2 × 10⁵ cells/well) were cultured in 24-well plates, and then cells were transfected with empty vector and/or expression vectors as indicated in the figure legends together with p-125luc reporter plasmid (100 ng/well) and an internal control vector, phRL-TK (Promega) (10 ng/well) using Lipofectamine 2000. The p-125luc reporter, in which the human IFNB1 promoter region (−125 to +1) is fused to luciferase gene, was provided by Dr. T. Taniguchi (University of Tokyo, Tokyo, Japan). The total amount of DNA (500 ng/well) was kept constant by adding empty plasmids.

Sf9 cells were seeded onto a 24-well plate (4 × 10⁵ cells/well) and were transiently co-transfected with p(-998/+71) (1 µg/well), pAC-AhR/pAC-ARNT/pAC-CncC (1 µg/well) and the internal Renilla luciferase control reporter plasmid phRL-TK (Promega; 0.2 µg/well) while using the Cellfectin-II reagent (Invitrogen; 2 µL per well). After 48 h, the cells were harvested, and the resulting lysates were used to measure the Renilla and firefly luciferase activities, as described above.