Dual luciferase reporter assay system

Liver tissue was pretreated with NP-40 lysate, the samples were dissolved in assay buffer and vortexed extensively for 2 min as described previously [27 (link)], and then, TG content was determined according to the manufacturer’s instruction (Abcam, Cambridge, UK). The luciferase activity assay was carried out using the Dual-Luciferase Reporter Assay System (GenePharma, Shanghai, China). HEK293T cells were cotransfected with miR-743a-3p mimic/inhibitor and GSTM1-3′-UTR reporter. Luciferase activity was evaluated 48 h after transfection, as described previously [28 (link)]. The coimmunoprecipitation (co-IP) assay was performed as Sun, et al. described [29 (link)]. In brief, cells were lysed in co-IP buffer on ice for 30 min. Then, the cells were centrifuged, and the supernatant was collected, followed by incubation with Flag Magnetic Beads (Sigma-Aldrich, St. Louis, MO) with gentle rocking overnight at 4 °C. The mixture pelleted was washed three times and then eluted in a loaded buffer and denatured at 95 °C for 10 min before western blotting. The reagents and methods for malonaldehyde (MDA), GST activity, mRNA and protein expression, immunohistochemistry, and immunofluorescence are detailed in Additional file 1.

WT or mutated BUB1B-promoter was cloned into the pGL3-Luc reporter vector (Genepharma, Shanghai). LUAD cell lines were plated into 24-well plates and transfected with ZNF143 plasmids using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) after 24 h. After 48 h, luciferase activity was measured by using the Dual-Luciferase reporter assay system(Genepharma, Shanghai) as the manufacturer’s instructions [20 (link)].

The full-length 3′-UTR of the endothelin A receptor (Ednra) gene was amplified by PCR and cloned downstream the pmirGLO Luciferase (Shanghai GenePharma Co., Ltd, China) multiple cloning site by digestion with the SpeI and HindIII restriction enzymes. The resulting construct was called pEdnra-wild-type (Wt). The miR-1929-3p binding site on the Ednra mRNA was mutated and cloned in the pmirGLO Luciferase plasmid to create the pEdnra-Mutant (Mut) vector. The Wt and Mut vectors were each transfected with and without miR-1929-3p into the HEK293 cells. Additionally, a dual-luciferase reporter gene assay was performed using the dual-luciferase reporter assay system (Shanghai GenePharma Co., Ltd, China).
The mmu-miR-1929-3p mimics, corresponding negative control and siRNA targeting Ednra were synthesized from GenePharma (Shanghai GenePharma Co., Ltd, China). Ednra sense 5′-CCTGGCAGGAAACAATGTCAAAGTGGCCAAATGAGCTGATTGTGTTAAGTGAGATGTAGTTACACC-3′ antisense 5′-TCGAGGTGTAACTACATCTCACTTAACACAATCAGCTCATTTGGCCACTTTGACATTGTTTCCTGCCAGGAGCT-3′; si-Ednra sense 5′-CGCAGAACAUCAAAGAAGATT-3′, antisense 5′-UCUUCUUUGAUGUUCUGCGTT-3′; negative control siRNA sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3′.