Renilla luciferase reporter

For the promoter activity assay, the cells were co-transfected with the proper amount of SOCS5: rs3814039_G/pGL4.17 or SOCS5: rs3814039_C/pGL4.17 together with pRL-SV40 Renilla luciferase reporter (Promega, for internal control) using TurboFect™ (Origene) at the basal level or induced by EGF or IL-6. At 48 hours post-transfection, both firefly and Renilla luciferase activity were determined by the Dual luciferase assay system (Promega) following the manufacturer's instructions. For the assay of miRNA target expression, the ESCC cells were transiently transfected with SOCS5: rs3768720_C/pmirGLO or SOCS5: rs3768720_A/pmirGLO using TurboFect™. Firefly and Renilla luciferase activity were both analyzed as described above.

Transcriptional activity assays of β -catenin/Tcf pathways were performed in the neural tube as described by (Alvarez-Medina et al., 2008). Chick embryos were electroporated at HH stage 11/12 with the following DNAs: Wnt1, FZD10, hLrp6 and FZD10 shRNA, or with empty pCA vector as control, together with a TOPFLASH luciferase reporter construct containing synthetic Tcf-binding sites [46 (link)] and a Renilla-luciferase reporter (Promega) for normalization. Embryos were harvested after 24 hours incubation and GFP-positive neural tubes were dissected and homogenized with a douncer in Passive Lysis Buffer. Firefly- and Renilla-luciferase activities were measured by the Dual Luciferase Reporter Assay System (Promega). Statistical analysis was performed by Student's t-test.

RIG-I signaling in Huh7, 293T and A549 cells with organelle-specific MAVS was measured indirectly by using dual luciferase reporter constructs as previously described [79 (link)]. Briefly, cells were seeded into 96 well plates 24 hours before co-transfection with either IFIT1, IFNB, or IFNL1 promotor constructs pGL3B or p125-Firefly-Luciferase (kindly provided by Ganes Sen, Cleveland and Takashi Fujita, Tokyo, respectively [79 (link), 80 (link)]). The co-transfected Renilla-Luciferase reporter plasmid pRL-SV40 (Promega, Germany) served as transfection control. Plasmid transfection was conducted with the Effecten transfection reagent (Qiagen, Germany) according to the instructions of the manufacturer. Cells were infected with different viruses 24 hours post transfection. For some experiments specified in the results section, cells were transfected with reporter plasmids 24 hours after lentiviral transduction and lysed 16 hours later. For mouse type I IFN bioassays, MEFs expressing organelle-specific MAVS variants were infected with either Sendai or Reo virus. Supernatant was harvested at given time points and stored at -80°C. Mouse L929-ISRE-Firefly-luciferase reporter cells [45 (link)] were seeded 24 hours prior to treatment. Reporter cells were stimulated with UV-inactivated supernatant and eight hours later firefly luciferase activity contained in cell lysates was determined.

HEK293 cells were seeded in 6-well plates (10⁵ cells/well) and transfected using Lipofectamine^® 2000 Transfection Reagent (Life Technologies), following the standard manufacturer’s protocol. Cells were transfected with 0.5 μg/well of the appropriate TBX15 promoter reporter construct and 0.25μg/well of renilla luciferase reporter (Promega) as internal control for transfection efficiency. After 24h of TNFα stimulation, cells were lysated in PLB buffer, and subsequently assayed for luciferase activity. When the expression of p65 was required, cells were cotransfected with promoter reporter construct and the pCDNA3.p65 expression plasmid (kindly provided by Dr. Leonardi, Naples, Italy) by using 0.15μg/well of luciferase reporter construct, 0.05μg/well of renilla reporter and 0.8μg/well of pCDNA3.p65 (or empty pcDNA3.1 as negative control). After 24h, cells were lysated and assayed for luciferase activity. Luciferase activity was measured using the Dual-Luciferase^® Reporter Assay System (Promega). All transfections were performed in duplicate, and experiments were repeated at least three times. The firefly luciferase activity was normalized according to the renilla luciferase activity.

A dual‐luciferase assay was performed to confirm the impact of YY1 on PRMT5 promoter activity. Briefly, cells on a 24 well plate were co‐transfected with the firefly luciferase reporter (50 ng) along with the Renilla luciferase reporter (Promega) (20 ng) for 24 hours using Lipofectamine 3000 according to the protocol provided by manufacturers. The luciferase activity was measured in cellular extracts using a Dual‐luciferase Reporter Assay Kit (Promega). The luciferase activity of each well was normalized to Renilla luciferase activity. The experiment was repeated in triplicate.

HEK293T cells were seeded in 24-well plates and transfected with ISRE-luc firefly luciferase reporter and Renilla luciferase reporter (Promega), together with the indicated combination of expression plasmids. At 24 h posttransfection, cells were transfected with plasmids encoding RIG-I for another 24 h or treated with IFNb for another 8 h. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (E1910; Promega, Madison, WI, USA) according to the manufacturer’s protocol using a GloMax 20/20 Luminometer (Promega).