X tremegene hp dna transfection reagent

For transient transfection, the cells were inoculated overnight and reached to 60–70 % confluence; a certain amount of plasmid DNA was transiently transfected into the cells with the X-tremeGENE HP DNA transfection reagent following the manufacturer’s protocol (Promega, Madison, USA). The cells were harvested 48 h post-transfection; they were subjected to the indicated experiments. To generate stable cell lines, the Changliver cells were transduced with lentiviral particles containing either TIPE2 shRNA or scramble shRNA. For ectopic expression of TIPE2, the Huh7 cells were infected with lentiviral particles expressing TIPE2 cDNA or empty vector. The cells were selected in 2 μg/ml puromycin for 2 weeks and were pooled for future study.

The truncated or mutated promoter sequences of the human RRM2 gene were amplified by PCR and inserted into the pGL3-Basic luciferase reporter vector (Promega, Madison, WI). Cells were plated onto 24-well plates the day before transfection. The cells were cotransfected with 0.5 μg of firefly luciferase reporter constructs, 0.02 μg of pRL-SV40 Renilla luciferase reporter plasmids (Promega), and 0.5μg Flag-CREB1 using the X-treme GENE HP DNA Transfection Reagent. The luciferase activity was measured by a dual-luciferase reporter assay system (Promega).