Prl cmv renilla luciferase reporter vector

H9c2 cells (0.5 × 10⁵ cells per well) were plated in 24-well culture dishes and maintained overnight at 37 °C with 5% CO₂. H9c2 cells were transiently co-transfected with PPARγ, ACO-PPRE and Renilla luciferase expression vector using Lipofectamine™ 2000 Reagent (Invitrogen, USA). After 6 h incubation with transfection mixtures, the culture medium was replaced by 10% FBS medium. Twenty-four hours after transfection, cells were treated with Honokiol (0, 2.5 μM, 5 μM) for one day. Finally, cells were harvested in a centrifuge tube and centrifuged at 1000 g for 5 minutes. The luciferase activity was measured in a luminometer using dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Transfection efficiency was normalized by Renilla luciferase reporter (pRL-CMV vector, Promega).

DNMT3B luciferase reporters containing DNMT3B wild-type 3′UTR (CUCUUCUUACUGGUGCUA) or mutant 3′UTR (CUCUUCUUACUCCUCCUA) were purchased from SWITCH Gear Genomics Company (Menlo Park, CA). MDA-MB-231 cells were co-transfected with the 50 ng luciferase reporter, 1 ng Renilla luciferase reporter (pRL-CMV vector, Promega, Madison, WI), or/and 100 nM mimic miR-29c by Lipofectamine 3000 (Invitrogen, Grand Island, NY), respectively. After transfection for 24 h, cells were lysed and centrifuged at 12000 rpm for 10 min. The supernatant was collected following centrifuging, and luciferase activities were measured using the Dual-Luciferase Assay System (Promega, Madison, WI). The activity of Renilla luciferase was normalized to that of firefly luciferase.

Full-length, linear monomeric HBV of specific D-subgenotype was released from pJET1.2/blunt vector by digestion with 1U of SapI/µg at 37 °C for 12 hours, which was followed by gel purification using QIAquick gel extraction kit (Qiagen). Huh7 cells, seeded into 12-well plates at a concentration of 2 × 10⁵ cells/well, were individually transfected with 1 µg/well of the HBV/D-monomers or pGL3-HBx constructs using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) along with pRL-CMV Renilla Luciferase Reporter Vector (Promega), which served as transfection normalization control. Six hours post-transfection, culture medium was replaced with fresh Dulbecco’s modified Eagle’s medium (DMEM) (Hi-Media Laboratories Pvt. Ltd., Maharashtra, India) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific). The values obtained from each experiment were normalized to Renilla luciferase levels. All experiments were performed in triplicates and repeated at least three times. The data was expressed in fold-change where the expression value of subgenotype-D1 was set at 1.0 and values of other D-subgenotypes were expressed relative to D1.