Dual luciferasetm reporter assay system

FuGENE 6 and Dual Luciferase^TM Reporter Assay System (Promega), Madison, WI, USA were used. The IL-8 promoter–luciferase reporter construct (pGL2-IL-8), AP-1 and NF-κB luciferase reporter plasmids, dominant-negative mutants p38 MAPK (pMCL-mP38), MEK-1 (pMCL-K97M), and JNK (pMCL-TAM67) were used as in our previous study [12 (link),16 (link)]. The transfection and co-transfection were performed as described previously [17 (link)]. Luciferase activity was determined using a luminometer (Centro XS LB960 microplate luminometer), Berthold Technologies, Bad Wildbad, Germany.

The binding sites between hsa_circ_0045881 and miR-214-3p were predicted via CircInteractome (https://circinteractome.nia.nih.gov/). miR-214-3p was cloned and inserted into a dual-luciferase reporter vector. MDA-MB-231 cells were seeded in 48-well plates, kept in a humid incubator for 24 h and then cotransfected with luciferase reporter plasmids and Renilla plasmids with Lipofectamine 2000 (Invitrogen, USA). The firefly and Renilla luciferase activities were analysed with the Dual-LuciferaseTM Reporter Assay System (Promega, USA) 48 h after transfection, according to the manufacturer’s instructions.

Cells were pre-seeded in a 24-well plate at a density of 1×10⁵ cells/dish. On the following day, the cells were co-transfected with 1μg of constructed promoter reporter plasmids or control plasmids and 200ng of pRL-TK plasmid by Lipofectamine3000 (Invitrogen, USA). Twenty-four hours after transfection, cells were collected. Firefly and Renilla luciferase activities were sequentially measured by a Dual-LuciferaseTM Reporter Assay system (Promega). Luciferase activity was normalized by Renilla activity for each well. All assays and analyses were carried out in triplicate.

Cells were stimulated as indicated and lysed for luciferase assay. Luciferase activity was measured by the Dual-Luciferase^TM reporter assay system according to the manufacturer's instructions (Promega Corp.).

Transient transfection of reporter plasmids and luciferase assays was performed as described previously^{4 (link), 80 (link)}. 1 × 10⁵ cells were seeded per well in 24-well plates 24 h before transfection. Lipofectamine^TM LTX and Plus^TM reagents (Invitrogen) were used for DNA transfection, according to the manufacturer’s instructions. 400 ng of pGL3p-N3Int2 was transfected along with or without 400 ng of pBABE-bla-ZEB1, pBABE-bla-ZEB2 of pBABE-bla (empty vector control). 5 ng of phRL-SV40-renilla luciferase vector (Promega) was co-transfected to calibrate the variation of transfection efficiencies among wells. Cells were incubated in the presence or absence of 1 µg/ml DOX to induce ICN1 in cells expressing ICN1^TetOn for 48 h before cell lysis. Alternatively, 5 ng/ml TGFβ1 was added at 24 h after transfection and incubated for an additional 48 h before cell lysis. Luciferase activities were determined using Dual-Luciferase^TM Reporter Assay system (Promega) and ORION Microplate Luminometer (Berthold Detection Systems, USA, Oak Ridge, TN). The mean of firefly luciferase activity was normalized with the co-transfected renilla luciferase activity. Transfection was carried out at least three times, and variation between experiments was not greater than 15%.

The artificially synthesized PEG3 3′-untranslated region (3′UTR), as well as a form with mutation of potential miR-129-5p binding sites, were mutated and introduced into the pGL3-reporter (Promega Corp., Madison, WI, United States). The wild-type (pGL3-PEG3-WT) or mutant type (Mut) (pGL3-PEG3-Mut) was co-transfected into primary HSCs (5 × 10³ cells/well) with miR-129-5p mimic or miR-NC, respectively. Cells were harvested and lysed at 48 h after transfection, and luciferase activity was measured using Dual-Luciferase^TM Reporter Assay System (Promega Corp., Madison, WI, United States) on a Luminometer TD-20/20 detector (E5311, Promega Corp., Madison, WI, United States). The target relationship between NEAT1 and miR-129-5p was verified similarly. In brief, NEAT1 WT or Mut was inserted into the pGL3-reporter to construct pGL3-NEAT1-WT or pGL3-NEAT1-Mut, which were then co-transfected with miR-129-5p mimic or miR-NC, respectively, into primary HSCs, and the luciferase activities were then determined.

VSMC were transfected using Lipofectamine LTX^TM and Plus Reagent (Invitrogen). cIAP2 constructs were co-transfected together with pCMV5/NOR-1 expression plasmid or the corresponding empty vector (pCMV5)52 (link). Luciferase activity was determined in cell lysates using the Dual-Luciferase^TM Reporter Assay System (Promega). Results were expressed as the ratio of firefly to renilla activity.