Prl sv

The Stat3-C Flag pRc/CMV (CA-STAT3) was a gift from Jim Darnell (Addgene plasmid #8722) [48 (link)], and pCDNA3.2/V5-DEST was from Thermo Fisher Scientific. pGL4.47[luc2P/SIE/Hygro] (a firefly luciferase reporter plasmid under the control of five copies of STAT3 responsive element) and pRL-SV (a Renilla luciferase reporter plasmid under the control of SV40 promoter) were provided by Promega. Huh7 cells in Opti-MEM medium (Thermo Fisher Scientific) were transiently transfected with pGL4.47[luc2P/SIE/Hygro] (300 ng) and pRL-SV (30 ng) for 6 h using a FuGENE^® HD Transfection Reagent (Promega). After replacing the transfection medium with the growth medium containing 10% FBS, the cells were treated with 5–20 μM HsA for 18 h. In some experiments, Huh7 cells were transfected with 300 ng of CA-STAT3 in conjunction with reporter plasmids for 12 h, and then exposed to HsA. The same amount of pCDNA3.2/V5-DEST was used for mock transfection. Luciferase activities in the cell lysates were determined using a Dual-Luciferase^® Reporter Assay System (Promega), and luminescence intensity of firefly luciferase was normalized by that of Renilla luciferase.

HeLa cells were cultured in GMEM with 10 % FCS. 10⁴ HeLa cells were seeded into a well of a 96-well plate. The following day, three wells of cells were transfected with 0.5 μg of the PiggyBac Sox expression vector, 0.5 μg of either SOX (AACAAAG) × 7 (tandem repeat)-tk-luc or SAC (CCGCGGT) × 7 (tandem repeat)-tk-luc and 10 ng of pRL-SV (Promega) followed by the culture for 24 h. The cells were then tested for luciferase activity using the Dual luciferase assay kit (Promega) with Centro LB 960 luminometer (Berthold).

Cells were seeded into 24‐well plates at a density of 2 × 10⁵ cells/well. And 24 h later, the cells were transfected with pNF‐κB‐TA‐luc (Beyotime) and marine luciferase pRLSV (Promega, Madison, Wisconsin, USA) according to Lipofectamine 3000 instructions (L3000008, Invitrogen) as internal standardized control. Twenty‐four h prior to transfection, cells were treated with ox‐HDL or lentivirus for 24 h, and then cotransfected with pNF‐κB‐TA‐luc and pRLSV. And 48 h after transfection, the luciferase activity was detected on a Luminometer TD‐20/20 detector (E5311; Promega) with the use of the dual luciferase reporter assay system kit (Promega).
The promoter region of FOS mRNA 3'UTR containing miR‐34a binding site was synthesized. The FOS 3'UTR‐wild‐type (WT) and FOS 3'UTR‐mutant (MUT) plasmids were constructed and introduced into pGL3‐REPORT™ miRNA expression reporter gene vector system (Promega). The above plasmids were then cotransfected with miR‐34a mimic and mimic NC, respectively. The luciferase activity was measured on Luminometer TD‐20/20 detector (E5311; Promega) with dual luciferase reporter assay system kit (Promega).