Human hek293t cell line

The human HEK293T cell line (American Type Culture Collection, ATCC, USA) was cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin in 5% CO₂ at 37 °C. All experiments were conducted when the culture plates achieved around 80% confluency. The cell line was tested to be free of Mycoplasma contamination.
To create a dusp1 knockout cell line, two sgRNAs targeting exon 1 and exon 2 of the human dusp1 gene were designed, synthesized, and cloned into PX458 plasmids (Addgene, USA), then transfected into the HEK293T cells with Lipofectamine (Thermo, USA). After screening for enhanced green fluorescent protein (EGFP) expression, monoclonal cells were established by serial dilution and culture, and those monoclonal cells with correct dusp1 knockout were identified by sequencing. The knockout efficiency of the cell lines was checked by western blot analysis. Cell lines in which the first and second exons of dusp1 were removed were chosen for further experiments.

The Human HepG2 cell line, mouse AML12 cell line, and human HEK293T cell line were procured from American Type Culture Collection. HepG2 cells and HEK293T cells were cultured in DMEM media (#10569044; Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (#16140071; Gibco) at 37°C with 5% CO₂. AML12 cells were cultured in DMEM/F12 media (#11320033; Gibco) containing 10% fetal bovine serum, 1 × ITS supplement (#PB180429; Procell, Wuhan, China), 40 ng/mL dexamethasone at 37°C with 5% CO₂. For in vitro experiments, HepG2 cells were treated with or without 20 μM of BBR in DMEM media containing 250 μM of PA for 24 h. AML12 cells were treated with or without 10 μM of BBR in DMEM/F12 media containing 200 μM of PA for 24 h. Mouse primary hepatocytes were extracted by using a two-step perfusion method using type IV collagenase [17 (link)]. Mouse primary hepatocytes were cultured in DMEM/F12 media containing 200 μM of PA and treated with or without 10 μM of BBR for 24 h. All control cells were supplemented with the same volume of dimethyl sulfoxide or bovine serum albumin to eliminate the influence of the solvent on the experiment. The BBR and PA concentrations in the cell experiments were chosen following CCK8 experiments and pertinent literature [7 (link)].