Dual luciferase reporting kit

The following two pmiR-RB-REPORT TM vectors were synthesized: Sox4 3ʹ-UTR with the putative target site of miR-30d (sox4 wt-3ʹ-utr); Sox4 3ʹ-UTR with mutation binding site (sox4 mut-3ʹ-UTR); 100ng of carrier (Sox4 WT-3ʹ-UTR, Sox4 Mut-3ʹ-UTR), miR-30d-mimics/inhibit and miR-NC (50 nM/well) were used together with Lipofectamine PTEN^® 2000 reagent (Invitrogen; Thermo Fisher Scientific) and transfected into 293T and SW1353. Analog control and Sox4 Mut-3ʹUTR were applied as NC. Fluorescein activity was tested by a dual luciferase reporting kit (Promega, Madison, WI, USA).

The human IL6 promoter region (Supplementary table 2) was cloned into the pGL6 firefly reporter vector. The vector was transfected into 293 T cells using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and cultured under normoxia (21% O₂) or hypoxia (1% O₂) conditions for 24 h. Cells were collected, lysed, and luciferase activity was measured using Dual‐Glo® Luciferase Assay System (Promega, Madison, WI, USA) in a Centro LB 960 Microplate Luminometer (Berthold).
Human B cells are transfected with PGLV6‐IL6‐promotor and PGLV6‐IL6p‐HBSdel plasmids using Thermo Fishers' Lipofectamine 3000 cell transfecting kit. The transfected B cells were then cultured for another 24 h in a 5% CO₂ 37°C incubator, with one plate placed in an Anaeropack to create a hypoxia situation while the other was placed in a normoxia environment before protein harvesting. Transfected human B cells were centrifuged at 4°C for 5 min at 450 g and then resuspended in RIPA lysis. After incubating on ice for 30 min, cell culturing plates were centrifuged at 4°C for 5 min at 1000 g. Cell lysis was transferred to a new black 96‐well plate, with 20 μL per well. The dual‐luciferase reporter assay was performed using Promega's dual‐luciferase reporting kit. A volume of 100 μL LAR was added to cell lysis for the first fluorescent signal measurement and 100 μL Stop&Glo reagent was added for the second measurement.