Dual luciferase report assay

The SNX5 promoter (−1,297 bp/–30 bp relative to the transcription start site) was cloned into the luciferase reporter gene vector pGL3-Basic (Promega). The fidelity of the constructs was confirmed by sequencing. The primer sequences are listed in Table S2.
Cells were cotransfected with the corresponding reporter plasmid and the indicated plasmids in each experiment according to the standard protocol. The pRL-TK reporter construct was used as the internal control. Luciferase activity was detected using a Dual-Luciferase Report Assay (Promega) system in accordance with the manufacturer's instructions.⁴⁴ (link)

The VPS35 promoter and 5′-truncated sequences of the VPS35 promoter were supplied by IGEbio (Guangzhou, China). The VPS35 promoter (bp −1626/+114 relative to the ATG start codon) was cloned into the luciferase reporter gene vector zx001. Two 5′-truncated sequences of the VPS35 promoter were generated by PCR and cloned into the luciferase reporter gene vector zx001. The fidelity of the constructs was confirmed by sequencing. The primer sequences are listed in Supplementary Table S2.
Cells were grown in 24-well culture plates and cotransfected with mixtures of the corresponding reporter plasmid and the indicated plasmids in each experiment according to the standard protocol. Luciferase activity was detected using a Dual-Luciferase Report Assay (Promega) system in accordance with the manufacturer’s instructions.

Agomir and antagomir of miR-430s and negative control were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences of three miR-430s used for microinjection were 5′-uaagugcuauuuguugggguag-3′ (miR-430a), 5′-aaagugcuaucaaguugggguag-3′ (miR-430b), and 5′-uaagugcuucucuuugggguug-3′ (miR-430c), respectively. MS-222 and L-glutamine were obtained from Amresco (Solon, OH, USA). Dulbecco’s modified Eagle’s medium (DMEM, high glucose) and fetal bovine serum (FBS) were obtained from Gibco/Invitrogen (Paisley, UK). Penicillin and streptomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine2000 and pmirGLO vector were obtained from Invitrogen (Carlsbad, CA, USA). A ClonExpress™ II One Step Cloning Kit was purchased from Vazyme (Piscataway, NJ, USA). A Dual-Luciferase Report assay was obtained from Promega (Madison, WI, USA). A TaKaRa PrimeSTAR^® HS DNA Polymerase kit was purchased from TaKaRa (Tokyo, Japan).

The INHBB promoter was cloned into the luciferase reporter gene vector pGL3-Basic. Three mutant sequences of INHBB promoter were amplified by PCR and were cloned into luciferase reporter gene vector pGL3-Basic. The RELA gene sequence was constructed on pCDNA3.1 (+) vector. The primer sequences used is listed in Supplementary Table S2. According to the Genomedtech protocol, cells were co-transfected with the corresponding reporter plasmid in each experiment. TK reporter constructs were used as internal controls. The Luciferase activity was detected using the dual-luciferase report assay (Promega) system.

Firefly and renilla luciferase activities were analyzed using the Dual-Luciferase Report assay (Promega) according to the manufacturer′s instructions. Luciferase activity values were obtained using a Turner Biosystems 20/20n single-tube luminometer. Renilla luciferase activity was first normalized to the firefly luciferase activity, and then this ratio was normalized to the control constructs noted for each experiment. At least four independent transfections for each condition were averaged. All experiments were performed in triplicates and measured at least three times.

LINC00152 promoter was subcloned into the pGL3-basic vector (Genechem, Shanghai, China). Then, pGL3-LINC00152 promoter vectors and sh-FOXM1#1 or sh-FOXM1#2 or FOXM1 or their corresponding NC were co-transfected into RA FLSs cells. Simultaneously, wild-type or mutant sequences of miR-1270 in LINC00152 (LINC00152-WT or LINC00152-MUT; Genepharma) were subcloned into the pmirGLO luciferase reporter vector and then co-transfected into RA FLSs with miR-1270 mimics or its NC. After transfection via Lipotransfectamine 3000, the Dual-Luciferase Report Assay (Promega, Madison, WI, U.S.A.) was experimented.