Fig. 4.

IFNγ induces MPC1/2 downregulation via stat3 activation. (A) After treated MC38 and CaCO-2 cells with PBS or IFNγ (50 ng/ml) for 48 h, the cell lysates were extracted and the levels of phosphorylation STAT1, phosphorylation STAT3 STAT1 and STAT3 were detected with western-blot, β-actin was used as internal control. (B-C) After treated MC38 and CaCO-2 cells with IFNγ (50 ng/ml) or IFNγ combined with Fludarabine (1 uM) or IFNγ combined with Stattic (5 uM) for 48 h. (B) The cell lysates were extracted and the levels of MPC1 and MPC2 were detected with western-blot. (C) 1.5 × 10⁴ of treated cells were seeded in 96-well plate in 100ul medium. 24 h later the supernants were collected and the level of lactate was detected. (D) After treated MC38 and CaCO-2 cells with IFNγ (50 ng/ml) or IFNγ combined with Stattic (5uM) for 48 h. the cells were trypsinized and stained with CellROX™ and detected the overall ROS level with flow cytometry. (E) After treated MC38 and CaCO-2 cells with IFNγ (50 ng/ml) or IFNγ combined with Stattic (10 uM) for 48 h. The cell lysates were extracted and the levels of full lenghth Caspase 7 and Caspase 3 and cleaved Caspase 7 and cleaved Caspase 3 were detected by western-blot and β-actin was used as internal control. In the same time, the cells were trypsinized and stained with Annexin-V and 7-AAD. The overall apoptosis was detected with flow cytometry. (F) Mice were inoculated with 5 × 10⁵ MC38 cells. When tumor size was 5 × 5 mm, mice were treated with PBS, IFNγ (10 μg/mouse), Stattic (10 mg/kg) or IFN-γ combined with Stattic intratumorally for 10 days. Tumor growth was measured (n = 6). Data shown are representative of three independent experiments and error bars represent mean ± s.e.m., N.S., no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test).