Dual luciferase reporter system

Polymerase activity was measured using a mini-genome assay as previously described (Song et al., 2011 (link)). Briefly, 293 T cells were co-transfected with a luciferase reporter plasmid p-Luci together with internal control Renilla plasmid and RNP genes (PDP2002-PB2, PDP2002-PB1, PDP2002-PA, and PDP2002-NP) either from MA H6 or from WT H6, and incubated for 48 h. The luciferase activity was measured using a dual-luciferase reporter system (Vazyme Biotech Co., Ltd.) according to the manufacturer’s instructions.

The potential miR-1301-3p binding site of circCYP24A1 and its mutant were synthesized and subcloned into pGLO-miR plasmids respectively, named as pGLO-circCYP24A1-wild-type (WT) and pGLO-circCYP24A1 mutant (MUT). As the same, the 3′-UTR of ALDH1A3 containing the predicted miR-1301-3p-binding sites or mutant sites was amplified and cloned into pGLO-miR, named as pGLO-ALDH1A3-WT and pGLO-ALDH1A3-MUT. HEK293T cells were co-transfected with indicated luciferase plasmids and mimics. Firefly luciferase activity and Renilla luciferase activity were determined after transfected 48 h with a Dual-Luciferase reporter system (Vazyme), and Renilla luciferase activity was set as internal control.

For cultivating 3T3-L1 and PK-15 cells, a total of 2 × 10⁵ cells were transferred to 6-well plates and transfected using transfection reagents to construct clones. Plasmids were transfected separately into the cultured 3T3-L1 and PK-15 cells, and after 48 h, the luciferase activity was evaluated using the dual-luciferase reporter system (Vazyme, Nanjing, China) as per the manufacturer’s instructions with a Modulus™ II Microplate Multimode Reader (Turner Biosystems, Sunnyvale, CA, USA). For each experiment, we repeated the process thrice and performed three independent experiments. PK15 and 3T3-L1 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. We maintained all the cells in a humidified atmosphere with 5% CO₂ in the air at 37 °C, and we purchased all the cell culture reagents from Thermo Fisher Scientific (Waltham, MA, USA). We processed the experimental results statistically using the SPSS17.0 software package (SPSS, Inc., Chicago, IL, USA) with the T-test, and we expressed the data as mean ± standard error.