Tofacitinib

HT-29.cl19A were obtained from Kim E. Barrett (University of California, San Diego, California), Caco-2BBe, A549 and THP-1 cells were originally obtained from ATCC and cultured according to the manufacturer’s recommendation in medium with 10% FCS. For PTPN2 knockdown, the cells were infected with lentiviral particles containing non-targeting control shRNA (Ctr) or PTPN2-specfic shRNA as described previously⁴⁹ and stable clones selected using puromycin. For STAT1 silencing, the cells were transfected with previously validated, STAT1-specific or non-targeting control siRNA constructs (Dharmacon) using DharmaFECT transfectionre agents as described previously^{50 (link)}. In experiments with STAT1 siRNA and IFN-γ treatment, the culture medium was replaced with serum-free medium 8 h prior to addition of IFN-γ (1000 IU; Roche). In experiments with tofacitinib, the cells were treated with tofacitinib (50 μM, MedChemExpress, Monmouth Junction, NJ). Control cells were treated with an equal amount of vehicle (dimethyl sulfoxide, DMSO, 0.5%, Sigma-Aldrich).

For the in vivo experiment, mice were treated through oral gavage with tofacitinib (40 mg/kg per day), idasanutlin (30 mg/kg per day) (MedChemExpress, New Jersey, USA), or vehicle (0.2% dimethylsulfoxide [DMSO]). The mice received medication daily until death. For the in vitro experiment, MOLT-4 cells were treated with tofacitinib (2.0 μM), idasanutlin (0.5 μM) or 0.2% DMSO in culture medium for 48 h and then proliferation and apoptosis were examined.