Dual luciferase reporter assay kit

BC cells in 24-well plates were co-transfected with the wild or mutant HREs reporter plasmids (Genecreate), together with HIF1α and renilla luciferase plasmids. 48 h later, the luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Hanbio) and calculated as the ratio of firefly to renilla luciferase activity.

StarBase predicted targeted binding sites for MALAT1 and miR-23b-3p and targeted binding sites for miR-23b-3p and α-synuclein. The miR-23b-3p and MALAT1 or α-synuclein 3′-UTR binding site and its mutated sequence were inserted into the pmirGLO dual luciferase vector (GenePharma, Shanghai, China). The constructed vector was cotransfected with miR-23b-3p mimic and its negative control (NC mimic) into cells using Lipofectamine 2000. Forty-eight hours after transfection, luciferase activity was detected using a dual luciferase reporter assay kit (Hanbio Biotechnology, Shanghai, China).

PTPN7 wild and mutant plasmids were constructed by the company (Hanbio, Shanghai; Supplementary table 3). HEK-293T cells, seeded in the 96-well plate, were co-transfected with PTPN7 plasmid and miR-592 mimics for 48 h. For the luciferase assay, the following working solutions were prepared: A: 10 μl (DMEM) + 0.8 μl (plasmid) + 0.25 μl (mimics/NC); B: 10 μl (DMEM) + 0.24 μl (lip2000). After mixing Solution A and Solution B, let the mixture stand for 10 min, then added it to each well and incubated for 48 h. Fluorescence expression was detected using a kit (Dual-Luciferase Reporter Assay Kit, Hanbio) under a chemiluminescent microplate reader (Thermo, VARIOSKANLUX).