Lipofectamine 2000 system

To further determine whether the activation of the Akt/Bad/Bcl-2 signaling pathway by LINC00473 is mediated by PEBP1, the shRNA and overexpression plasmid of PEBP1 were transfected into Dex-induced hBMSCs using a Lipofectamine 2000 system (Thermo Fisher Scientific, Inc.), as recommended by the manufacturer. Three shRNAs of PEBP1, including sh-PEBP1-1 (target sequence, tgGGA TGA CTA TGT GCC CAA A), sh-PEBP1-2 (target sequence, caAAT TCA AGG TGG CGT CCT T) and sh-PEBP1-3 (target sequence, cgAGC AGG ACA GGC CGC TAA A), and the overexpression plasmid (vector name, GV657) for PEBP1 (PEBP1-up) were provided by Genechem Corporation. Moreover, a random sequence (TTC TCC GAA CGT GTC ACG T) was selected as the negative control of shRNA (sh-Control), and a blank vector was used as the negative control of the overexpression plasmid for PEBP1 (PEBP1-Control).
Following infection with the LINC00473-up lentivirus, the Dex-induced hBMSCs were transfected with a shRNA of PEBP1 to create the Dex + LINC00473-up + sh-PEBP1 group (Dex + LINC00473-up + sh-PEBP1). The Dex-stimulated hBMSCs were transfected with the recombinant plasmid of PEBP1 to create the Dex + PEBP1-up group (Dex + PEBP1-up). Following transfection for 24 h, the transfection efficiency of PEBP1 was detected by RT-qPCR and western blot analysis.

The pMIR-REPORT System (Thermo Fisher Scientific) was used to identify miR-467b-3p binding sites in the 3ʹUTR of Gpat1. The β-galactosidase reporter control vector was used for normalization. The putative or mutated miR-467b-3p binding sites of Gpat1 3ʹUTR were cloned into a luciferase miRNA expression reporter vector ,which contains firefly luciferase under the control of a mammalian promoter/terminator system. Hepa1–6 cells were co-transfected with the Gpat1 3ʹUTR luciferase reporter and the control construct, and with the miR-467b-3p mimic or mimic control, using the Lipofectamine 2000 system (Thermo Fisher Scientific). After 48 hours of transfection, luciferase activity was measured using the Dual-Light System (Applied Biosystems) and normalized to the corresponding β-galactosidase activity.
For the measurement of HNF4α signaling, Hepa1–6 cells were transfected with negative control or inducible HNF4α reporter plasmid from the Cignal HNF4α Reporter Assay Kit (SABiosciences, Hilden, Germany), along with a constitutively expressed Renilla construct. After 24 hours of transfection, cells were treated with 6-G for 48 hours. Luciferase activity was measured using the Dual-Light System.

HepaRG cells were transfected with pcDNA3-CBR1 wild type (CBR1/WT) or pcDNA3-basic using the lipofectamine 2000 system (Thermo Fisher Scientific). Stable cell lines were established as described previously (Tak et al., 2011 (link)). Briefly, the transfected HepaRG cells were maintained in a culture medium treated with G418 disulfate salt (600 mg/ml, A1720; Sigma-Aldrich), and G-418 resistant colonies were selected.