Pifithrin α pft α

The human hepatoma cell lines HepG2, Hep3B and the human embryonic kidney cell line HEK293T were purchased from ATCC cell bank in 2014. The human hepatoma cell line Huh7 was purchased from the cell bank of the Shanghai Institute of Cell Biology (Shanghai, China) in 2017. HepG2 and Hep3B were maintained in Minimum Essential Medium (MEM, Gibco, Thermo, USA). Huh7 and 293T were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo, USA). The complete cell culture mediums were supplied with 10% fetal bovine serum (FBS, Excel, Australia), 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were incubated in a CO₂ incubator at 37 °C, with 5% CO₂. All the cell lines were used for less than 25 passages from acquisition to discard. Pifithrin-α (PFT-α) was purchased from Santa Cruz Biotechnology (USA), nutlin-3 was from Selleck Chemicals (USA) and TPEN was from Sigma-Aldrich Inc. (USA). For the inhibitory assay, PFT-a (20 μM), nutlin-3 (50 nM) and TPEN (100 nM) were dissolved in DMSO, Milli-Q sterile water or ethanol, respectively, and applied to pretreat the cells.

Cell apoptosis was detected by fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI) Apoptosis Detection Kit (556547, BD Biosciences, USA) according to the manufacturer’s instructions. Briefly, stably infected KGN cells were cultured in the medium supplemented with either 20 µM Pifithrin-α (PFT-α, Santa Cruz Biotech), 10 mM N-acetyl-l-cysteine, or neither. They were harvested after 6 h incubation, washed with ice-cold PBS, resuspended in 500 μl 1 × binding buffer supplemented with 5 μl Annexin-V-FITC and 1 μl PI for 30 min, and left in the dark at RT. Flow cytometric analysis was performed on a BD FACS Aria Fusion (BD Biosciences). Data acquisition and analysis were performed using FlowJo V8 software (TreeStar, USA).

Pifithrin-α (PFT-α, Santa Cruz, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma, USA) to a final stock concentration of 20 µmol/l.