Dual luciferase system kit

We used two different ISRE-luciferase reporter assays: (i) an assay using the ISRE3 reporter plasmid (pGL4.10[luc2] backbone, Promega #E6651), which contains, as previously described^{158 (link)}, three repeats of the ISRE sequence (5’-GGAAAGGGAAACCGAAACTGAA-3’) separated by spacers designed on the basis of the ISRE motif from the ISG15 promoter, (ii) an assay using the ISRE5 reporter plasmid, which contains, as previously described^{139 (link)}, five repeats of the ISRE sequence 5’-GGGAAAGTGAAACTA-3’. HEK293T cells were transiently transfected, in 96-well plates, with the (ISRE) reporter plasmid (100 ng/well and 100 μL DMEM-10% FBS medium), the pRL-SV40 vector (Promega, #E2231, 40 ng/well) and the IRF1 WT or mutant p.CMV6 plasmid (100 ng/well), with the Lipofectamine LTX kit (Thermo Fisher Scientific, #15338–100), according to the manufacturer’s instructions. Cells were used for the ISRE assay with the Dual-Luciferase system kit (Promega #E1980), according to the manufacturer’s protocol, 24 hours after transfection. Signal intensity was determined with a Victor X4 plate reader (Perkin Elmer). Experiments were performed in triplicate, and dual reporter activity is expressed as the fold-induction relative to cells transfected with the empty vector.

The full length mRNA sequence of TUSC3 (3888 bp, NCBI Reference Sequence: NM_006765.3) was synthesised and then inserted into the HindΙΙΙ and MluΙ restriction sites downstream of the luciferase open reading frame of the pMIR-REPORT luciferase vector (Ambion, Carlsbad, CA, USA). For sequence point mutation, site-directed mutagenesis of the potential target site in the TUSC3 mRNA was performed using the QuikChange Site-Directed Mutagenesis kit (Promega, Madison, WI, USA). The correct clones were confirmed by sequencing. HEK 293 and GBM cells were seeded into a 24-well plate (5 × 10⁴ per well) and co-transfected with miR-UL112-3p mimics/inhibitor (50 nM) and the wild-type/mutant pMIR-REPORT luciferase vector (200 ng) using Lipofectamine^TM 2000 reagent according to the manufacturer’s instructions. Luciferase activity was detected 48 h after co-transfection using the dual luciferase system kit (Promega, Madison, WI, USA). The relative luciferase activity was calculated by normalising firefly luminescence to that of renilla.

We used the ISRE3 reporter plasmid (pGL4. 10[luc2] backbone, Promega #E6651), as previously described [25 (link)]. HEK293T cells in 96-well plates were transiently transfected with the (ISRE) reporter plasmid (100 ng/well and 100 µL of DMEM-10% FBS medium) and the pRL-SV40 plasmid (Promega, #E2231, 40 ng/well), with or without the IRF1 WT p.CMV6 plasmid (50 ng/well), with or without IRF8-WT or mutant forms, in the presence of the Lipofectamine LTX kit (Thermo Fisher Scientific, #15,338–100) and with or without EV, to ensure that the same amount of plasmid was present in each well, in accordance with the manufacturer’s instructions. Cells were used for the ISRE assay with the Dual-Luciferase system kit (Promega #E1980), according to the manufacturer’s protocol, 24 h after transfection. Signal intensity was determined with a Victor X4 plate reader (Perkin Elmer). Experiments were performed in triplicate, and results are expressed as dual reporter activity.

The TBX1 3′-UTR wild-type (WT) and mutated (MUT) sequences of the target gene, TBX1, were cloned into the pSICheck-2 plasmid. Next, 293 T cells were incubated into 96-well plates. Objective plasmids and mutant plasmids were mixed with miRNA mimic and scrambled RNA as a negative control, respectively. Lipofectamine 2000 transfection reagent was added. Cells were collected after 48 h of transfection. The manufacturer's instructions were followed for the Dual-Luciferase System Kit (Promega, USA).