Stop and glo reagent

The 293T cells were grown on 96-well plates. At 60–70% confluence, the cells were co-transfected with a mixture of the plasmids (50 ng per plasmid) pPB1, pPB2, pPA, pNP, pPolPR8MLuc and the respective PB1-F2 expressing plasmids (pPB1-F2 PR8/pPB1-F2 PR8 3KR/pPB1-F2 HK97/pPB1-F2 HK97 4KR/pPB1-F2 Stop3aa). Turbofect in vitro transfection reagent (Thermo Scientific) was according to the manufacturer’s instruction. At 48 hr p.t., the cells were gently washed with PBS and directly lysed in passive lysis buffer (Promega). Twenty microliters of the supernatant was transferred to an opaque, flat-bottom 96-well plate (Costar). A Synergy HT Multi-Mode Microplate Reader with automatic dispensers was used to add 50 μl LarII reagent (Promega). After a 2-second delay, the firefly luciferase luminescence in separate wells was measured. Subsequently, 50 μl of the Stop and Glo reagent (Promega) was added, and after a 2-second delay, the renilla luciferase luminescence in separate wells was measured. Three independent experiments were performed in triplicate for each sample. The luminescent signal for firefly luciferase was normalized to the renilla luciferase luminescent signal. The results were further normalized to the activity of vRdRp in the presence of the wt PB1-F2. The error bars indicate the SD for all independent measurements.

Cells were transiently transfected with 2 μg of firefly/renilla luciferase reporter plasmid. Protein extracts were prepared using the Passive Lysis Buffer provided in the Dual-Luciferase Assay (Promega). Equal amounts of protein extracts were plated into a 96-well plate. Firefly luciferase activity was measured for 12 seconds using the LB 960 luminometer (Berthold Technologies, Thoiry, France). To assess the internal standard activity, Stop and Glo reagent was added (Promega), and the peak of the renilla luciferase activity was then measured. Normalized relative luciferase units (RLU) were then calculated as firefly luciferase units of protein extracts of treated or untreated cells divided by renilla luciferase units of protein extracts of untreated cells. Data represent the mean ± SEM of three independent experiments, each performed in duplicate.

The seeding of HEK293T cells took place into 96-well plates and underwent culture to 50–70% confluence prior to transfection. For circRBMS3 and miR-424, 600 ng plasmids of circRBMS3-wt and circRBMS3-mut, 20 nmol miR-424 and N.C. were transfected. Following incubation for 48 hours, the detection of firefly and Renilla luciferase activities was conducted by the Promega Dual-Luciferase system. For providing an internal reference, firefly luciferase activities were evaluated with a 100 ml Luciferase Assay Reagent II (LAR II) (Luciferase Assay Reagent, Promega), and then 20 ml lysis buffer, moreover 100 ml Stop and Glo® Reagent (Luciferase Assay Reagent, Promega) was utilized for evaluating Renilla luciferase activities. Calculation of the difference between a firefly and Renilla luciferase activities was to evaluate relative luciferase activity.