Dual luciferase reporter assay reagent

The human prostate cancer cell lines LnCap, DU145 and PC3 cell lines (obtained from the Duke Cell Culture Facility, Duke University, Durham, NC, USA) were maintained in RPMI (Roswell Park Memorial Institute Medium) with 10% fetal bovine serum (Invitrogen, Waltham, MA, USA). The c-Jun siRNAs (SI00034678 and SI00300580), non-targeting siRNA control, and c-Jun primer were obtained from Qiagen (Hilden, Germany). The Dual Luciferase Reporter Assay reagents were from Promega (Madison, WI, USA), and Jet prime reagent was from Polyplus (Illkirch, France). Matrigel was obtained from BD Biosciences (San Jose, CA, USA)). c-Jun Chip-grade antibody for ChIP assay was from Abcam (Cambridge, UK), c-Jun antibody for western blot was from Cell Signaling Technology (Danvers, MA, USA) the GNA12 antibody was from Gentex (Irvine, CA, USA), and GNA13 antibody was from Calbiochem (San Diego, CA, USA), α-Tubulin was purchased from Sigma (St. Louis, MO, USA), protease inhibitors were purchased from Roche Applied Science (Penzberg, Germany).

To prepare the enhancer-reporter constructs, the Satb1 enhancer sequence was cloned into the KpnI and XhoI sites of pGL4.25 [luc2CP/minP] firefly luciferase vector containing a minimal TATA promoter (Promega). Rcho1 TS cells were used for the reporter assay. Twenty-four h after plating in 12-well plates, Rcho1 cells were transfected with the enhancer-reporter vector along with a control Renilla luciferase vector (pGL4.74 [hRluc/TK]) using Lipofectamine 2,000 (Thermo Fisher Scientific). Expression vectors for SATB1, SATB2 or ELF5 were individually cotransfected with the reporter vector to assess their regulatory role on the enhancer sequence. 12 h after the transfection, transfection medium was replaced with cell proliferation medium and cultured for another 12 h. 24 h after transfection, cells were washed with cold PBS, lysed in 100 µl of passive lysis buffer and standard dual luciferase assays were performed on the cell lysates by using Dual-Luciferase Reporter Assay reagents (Promega).
ChIP assays- ChIP assays were performed as described above.

To study IL-6 transcription, RAW 264.7 cells were transiently transfected with p1168huIL6P-luc+ plasmids (Belgian Co-ordinated Collections of Micro-organisms, BCCM^TM, Brussels, Belgium) (1 μg/10⁶ cells) with transfection reagent FuGENE HD (Promega Corporation, Madison, WI). Cells were cotransfected with the Renilla luciferase reporter plasmid pRL-TK (Promega) (50 ng/10⁶ cells) as an internal control. Twenty-four hours after transfection, the cells were stimulated with LPS, PA or both LPS and PA. To study nuclear factor kappa B (NFκB)-mediated transcription, RAW264.7 cells were transfected with 1 μg of NFκB Cignal Reporter (Qiagen Inc., Valencia, CA) using FuGENE^® HD as transfection reagent for 24 h. The renilla luciferase constructs were used as control. The cells were then treated with fresh medium containing 1 ng/ml of LPS, 100 μM of PA or LPS plus PA for 12 or 24 h. After the treatment, the cells were rinsed with cold PBS and lysed with the buffer from Dual-Luciferase Reporter Assay System (Promega). Both firefly and renilla luciferase levels were assayed in a luminometer using the dual-luciferase reporter assay reagents (Promega) according to the instruction from the manufacturer. The firefly luciferase levels were normalized to the renilla luciferase levels.

Different fragments of the GASA6 (1.4, 1.2, 1.1, or 0.9 kb) promoter were each cloned into the pGreen II 0800-LUC vector (Hellens et al., 2005 (link)) to generate reporter constructs. Full-length HBI1, BEE2, or IBH1 cDNAs were cloned into the pBlueScript vector with the Cauliflower mosaic virus (CaMV) 35S promoter and rbcS terminator to generate effector constructs. Each reporter construct, together with either 35S::HBI1, 35S::BEE2, or 35S::IBH1, was co-transformed into the mesophyll protoplasts of A. thaliana for transcriptional activity assay.
Single or double mutants of the GASA6 (1.4 kb) promoter were generated with the MutanBEST Kit (TAKARA, Japan) and subsequently cloned into the pGreen II 0800- LUC vector. Firefly and Renilla luciferase activities were assayed with the microplate luminometer (Turner Biosystems, USA) and the Dual-Luciferase Reporter Assay reagents (Promega, USA). Primers used for plasmid construction for protoplast assay are listed in Supplementary Table S1.

BHK/T7-9 cells (1 × 10⁵ cells/well) were seeded in a 12-well plate and incubated for 16 h in a 5% CO₂ incubator. The cells were transfected with either pCAGGS-SFTSV N or pCAGGS-SFTSV mt-N together with pCAGGS-SFTSV L, pATX-vRNA-Rluc, and a firefly luciferase expression plasmid for the standardized measurement of gene transfer^{37 (link)}. The pCAGGS/MCS plasmid without any insertion was used as a negative control. All transfected cells were incubated at 37 °C in a 5% CO₂ incubator for 24 h. The culture supernatant of each well was removed, and the cell pellet was lysed using 20 µL of passive lysis buffer (Promega Co., Madison, WI, USA). The cell lysate was reacted with Dual-Luciferase Reporter assay reagents (Promega Co.), and the Renilla luciferase and firefly luciferase activities were measured using the GloMax-Multi Detection System (Promega Co.). Polymerase activity was determined based on the ratio of Renilla luciferase activity to firefly luciferase activity.
All data were presented as mean ± standard deviation. Statistical analysis was performed using a one-tailed Student’s t-test. A P-value of > 0.01 was considered significant.