Prl cmv renilla luciferase reporter plasmid

The allele-dependent promoter activity of CHI3L1 was detected by the dual-luciferase reporter gene system. HEK293 cells were transfected with the Firefly luciferase reporter plasmid pGL3-basic (Promega, USA) under the control of the CHI3L1 promoter region containing each allele of rs10399931. The pRL-CMV renilla luciferase reporter plasmid (Promega, USA) was cotransfected for normalization of transfection efficiency, and Dual-Luciferase reporter assays were read 24 h later with a GloMax 96 Microplate Luminometer. RNAs of 52 subjects were extracted from the whole blood stored in Tempus™ Blood RNA Tubes by Terpus™ Spin RNA Isolation Reagent Kit (Thermo Fisher Scientific, USA), then reverse transcribed to cDNA by PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Japan). Real-time quantitative PCR was applied to detect the relative mRNA expression with different genotypes of CHI3L1 rs10399931 with QuantiNova™ SYBR Green PCR Kit (QIAGEN, Germany).

HEK293-EcI cells were transfected 2 days after seeding at a density of 3 × 10⁵ cells/mL in 6-well plates in DMEM with 4.5 g/L glucose, L-glutamine, and sodium pyruvate with 10% HI FBS, and 1% penicillin-streptomycin. The culture medium was refreshed on the day of transfection. Transfection was performed using Lipofectamine 3000 following the manufacturer’s instructions. For all experiments, 1.5 μg/well of pERV3 receptor plasmid (Agilent Technologies, 217468) was transfected along with 750 ng/well of pEGSH-LUC luciferase reporter plasmid (Agilent Technologies, 217468), and 250 ng/well of pRL-CMV Renilla luciferase reporter plasmid (Promega, E2261) as a reference. After 2 days of incubation in the conditions described above, the cells were washed twice with PBS and then transferred to a clear 96-well plate in DMEM with 4.5 g/L glucose, sodium pyruvate; without L-glutamine, phenol red at 100 μL/well. Final concentrations ranging from 300 to 300 μM of 20E or CF were added immediately after, with a final volume of 125 μL/well. The treated cells were then incubated for 1 day in the same conditions. The Dual-Luciferase Reporter Assay was performed as described previously (Okamoto et al., 2018 (link)).

The bim-luciferase (Luc) reporter, containing a 5.2 kb DNA fragment, was described previously.^{29 (link), 30 (link)} For the dual-luciferase reporter assays, cells were transfected with 1 μg of a luciferase reporter plasmid and 200 ng of the pRL–CMV Renilla luciferase reporter plasmid (Promega, Beijing, China). To test the effect of CPTH2 on the reporter, neurons were kept in conditioned media for 12 h after transfection and then transferred to serum-free media 25K containing CPTH2 (50 μM) for 24 h. To observe the effect of siGCN5s on the reporter, neurons were kept in conditioned media for 48 h after transfection. Firefly luciferase activity was normalized to Renilla luciferase activity as reported previously.^{37 (link)}