Dual glow kit

293T cells stably overexpressing TLR2 (TLR2-293) and TLR4 (TLR4-293) were seeded on 96-well plates (NEST, #701101, China) and transfected with NFκB-Luc (Firefly luciferase, experimental reporter, 100 ng/well) and pRL-TK reporter (Renilla luciferase, internal control, 5 ng/well) plasmids (Clontech, USA), respectively. 24 h post-transfection, cells were pre-incubated with DsCystatin (4 µM) or GST (4 µM) for 2 h and then stimulated with LPS (200 ng/ml, Sigma, #L2630, USA) or B. burgdorferi spirochetes (MOI = 1.0). The luciferase activity was measured after another 24 h, using a Dual Glow kit according to the manufacturer’s instructions (Promega, #E2920, USA).
To assess the effect of DsCystatin on distinct molecules in TLR4 pathway, TLR4-293 cells were seeded on 96-well plates and transfected with NFκB-Luc, pRL-TK, together with MYD88, TRAF6, NEMO, IKKα, or NFκBp65 plasmids, respectively. After 24 h post-transfection, cells were incubated with DsCystatin (4 µM) and GST (4 µM) for 24 h. The activation of NFκB induced by overexpression of distinct signal molecule was detected by luciferase assay as described above.

For luciferase reporter assays, 70% confluent HEK293T (or A549) cells were transfected with 10 ng of pRL-TK reporter (herpes simplex virus thymidine kinase promoter driving renilla luciferase; internal control), 100 ng of NFκB or AP1 luciferase reporter (firefly luciferase; experimental reporter) plasmid, and either 100 ng of recombinant expressing plasmids (Vector, MMP3-flag or MMP3 fragments) or 50 nM siRNAs (N.C. or MMP3 siRNA). At 24 h post-transfection, cells were infected with DENV-2 at a MOI = 1. The luciferase activity was measured after another 24 hrs, using a Promega Dual Glow kit according to the manufacturer's instructions.

293T cells that expressed IL-1α propiece or venus as control were cultured in 100mm dish to reach 80% confluent. The cells were transfected with 10 ug of pRL renilla luciferase reporter plasmid as internal control and 10ug NFκB luciferase reporter plasmid. At 48 hours post-transfection, the luciferase activity was measured using a dual glow kit (Promega, Fitchburg, WI) according to the manufacturer's instruction. Briefly, discard growth media from cultured cells then wash one time by 1×PBS. Lyse cells for 15 minutes at room temperature by 1×Passive Lysis Buffer (PLB). Transfer 20 μl cell lysate to a new plate and measure the firefly luciferase activity after dispensing 100 μl Luciferase Assay Substrate in Luciferase Assay Buffer II. Then measure Renilla luciferase activity after adding 100μl of Stop & Glo® Reagent.
Progressive deletions were made in the SP1 promoter region and inserted between the NheI and XhoI sites of the reporter luciferase vector pGL3-basic (Promega, Fitchburg, WI). The primers used for constructing the SP1 region are listed in Supplementary Table 3. SP1-1, SP1-2, SP1-3 stand for 1612 bp, 443 bp, 146 bp from the 5’-UTR of the sequence to the translational start site.

IFNβ- or ISRE-Luciferase reporter assay was performed as described previously[39 (link),43 (link)]. Briefly, 293T cells were transfected with IFNβ- Luc (or ISRE-Luc) (Firefly luciferase, experimental reporter, 100 ng/well) and pRL-TK reporter (Renilla luciferase, internal control, 5 ng/well) plasmids (Clontech, USA), IFNβ activator RIG-I-N (the active caspase recruitment domain (CARD) containing form of RIG-I), together with individual NS proteins from DENV1 A-E or vector control. (For ISRE-Luc assay, cells were also treated with IFNα (1000 U/ml) for 6h instead of stimulation with RIG-I-N transfection.) 24 h post-transfection, cells were lysed and the luciferase activity was measured using a Dual Glow kit according to the manufacturer’s instructions (Promega, USA).

For luciferase reporter assays, 70% confluent HEK293T cells were transfected with 10 ng of pRL-TK reporter plasmid (herpes simplex virus thymidine kinase promoter driving Renilla luciferase, internal control), 100 ng of IFNβ luciferase reporter plasmid (firefly luciferase, experimental reporter), 50 ng of IFNβ activators (RIG-IN, MAVS, TBK1, IKKε, IKKα, NFκB, or IRF3), as well as either 100 ng of recombinant expressing plasmids or siRNAs [Vector, DDX25, 50 nM negative control (N.C.), or DDX25 siRNA]. For measuring the activation of transcription factor NFκB and IRF3, NFκB and IRF3 responsive element specific reporter plasmids were used in the luciferase reporter assays. Subconfluent HEK293T cells were transfected with 10 ng of pRL-TK reporter, 100 ng of NFκB (pNFκB-Luc), or IRF3 (pPRD(III–I)-Luc) luciferase reporter plasmid, various doses of recombinant expression plasmids (Vector or DDX25), along with 50 ng of expression plasmids of RIG-IN. At 24 h post-transfection, cells were infected with DENV-2 at an MOI of 1 and incubated further for 24 h. Luciferase activity was measured using a Promega Dual Glow Kit according to the instructions of the manufacturer (Promega, USA).

In a 24-well culture plate, 70% confluent HEK293T cells were transfected with 50 ng of pRL-TK reporter (herpes simplex virus thymidine kinase promoter driven renilla luciferase; internal control), 100 ng of ISRE-luciferase reporter (firefly luciferase; experimental reporter) plasmid, and 100 ng of each Zika gene expression plasmid using Lipofectamine 2000. The cells were incubated at 37°C, 5% CO₂ for 24 h, and then transfected with 10 μg/ml of polyI:C which induces cellular RLR signaling and subsequent IFN-I production. The luciferase activity was measured 16 h after polyI:C stimulation using a Promega Dual Glow kit according to the manufacturer’s instructions.