Renilla glo

HEK293T-Rex cells incorporating either the ERSE-FLuc or XBP1s-RLuc reporters were plated at 20 μL/well from 250,000 cells/mL in white 384-well plates (Corning). Cell plates were centrifuged for 1 min at 1000 rpm, then incubated at 37°C overnight. The following day cells were treated as described with various concentrations of Thapsigargin or Tunicamycin, incubated for a further 18 hr at 37°C, equilibrated to room temperature, then 20 μL of SteadyLite or Renilla-Glo (Promega, Madison, WI) were added to each well. Luminescence activity was measured 10 min after reagent addition with an EnVision Multilabel Reader (PerkinElmer) using a 100 ms integration time.

RNAi suppression assays were performed in both Aag2 and S2 cells. Aag2 cells (1.7 × 10⁵ per well in a 24-well plate) were infected with RVFV MP12 or the positive-control CYV at an MOI of 5 and incubated for 24 h at 28°C. Cells were then cotransfected with 100 ng pIZ-Fluc (64 (link), 65 (link)), 40 ng pAct-Renilla (66 (link)) (FLuc and RLuc expression plasmids, respectively), and 0.05 ng dsRNA targeting FLuc or a control dsRNA targeting eGFP using Lipofectamine 2000. At 24 h p.t., cells were lysed in passive lysis buffer and FLuc and RLuc expression was determined using Bright-Glo and Renilla-Glo substrates (Promega).
RNAi suppression assays in S2 cells have previously been described (67 (link), 68 (link)). Briefly, 5 × 10⁴ S2 cells were seeded in 96-well plates and left to adhere overnight. Cells were transfected with 50 ng pMT-Fluc and 15 ng pMT-Rluc for 24 h at 28°C, after which the cells were either infected with RVFV MP12 at an MOI of 5 for 24 h or left uninfected. A 25-ng volume of dsRNA targeting either FLuc or eGFP as a control was fed to the cells. At 7 h after the feeding of dsRNA, expression of luciferase reporters was induced by addition of 0.5 mM CuSO₄ per well. After 24 h, cells were lysed and luciferase expression was determined as described above.

293T cells were treated with 500 μM DFMO for 2 days and subsequently transfected using Lipofectamine 2000 (Life Technologies) with CHIKV replicon RNA (32 (link)). For luciferase assays, cells were combined with Renilla (Renilla-Glo; Promega) or firefly (Bright-Glo; Promega) luciferase substrate at 24 h posttransfection. Luciferase assays were performed according to the manufacturer's recommendations (Promega), and results were measured using the Wallac 1420 instrument (PerkinElmer).

HEK293T and U87-MG cells were co-transfected with hRuc reporter and FLuc control plasmids using Lipofectamine 2000 for 24 h. Following cell lysis with Passive Lysis Buffer (Promega) for 20 min according to the manufacturer’s protocol, Renilla and firefly luciferase activities were determined using Renilla Glo and Luciferase Assay Systems (Promega), respectively, using a Wallac Victor3 1420 multilabel counter system and Wallac 1420 Manager v3 (Perkin Elmer), or Spectramax i3X system and SoftMax Pro v6.5.1 (Molecular Devices).

Renilla luciferase mRNA was transcribed from pRL-CMV (Promega) linearised with Xba1. Dicistronic RHRVF mRNA was transcribed from pRHRVF6 (link) linearised with BamH1. In vitro transcription for in vitro translation and UV crosslinking assays was carried out using a T7 maxiscript kit (Ambion) with or without cap analogue to generate capped or uncapped mRNA, followed by use of a polyadenylation kit (Ambion) if polyadenylated mRNA was required. Generation of capped and polyadenylated RHRVF mRNA for transfection into mammalian cells was carried out using a T7 mMESSAGE mMACHINE and polyadenylation kit (Ambion). In vitro translation assays were carried out in Flexi rabbit reticulocyte lysate (Promega) programmed with 20 ng/μl mRNA, with the addition of 0.5 mM MgCl₂ and 60 mM KCl. The reactions were supplemented with 20 ng/μl recombinant Unr, Unr mutants, or H100 buffer as a control. Unr was in 3.7 fold molar excess over mRNA. Luciferase assays were carried out using the Renilla-Glo or dual luciferase assay systems (Promega).