Figure 4

IFNγ-induced JAK2/STAT1/IRF-1/PD-L1 signaling is inhibited with AZD1480 (an ATP-competitive JAK2 inhibitor) and fludarabine (a STAT1 inhibitor depleting STAT1 mRNA and protein) in EBV (+) GC cells. (A) EBV (+) SNU-719 cells were incubated with increasing concentrations of AZD1480 or fludarabine (5, 10, and 20 μM) for 2 h, and then transfected with the PD-L1 promoter luciferase vector. After 4 h, cells were stimulated with 10 ng/mL IFNγ for 24 h, luciferase activities were measured. (B) EBV (+) SNU-719 cells were treated with 5 μM of AZD1480 or fludarabine for 2 h, followed by stimulation with 10 ng/mL IFNγ for 24 h. The mRNA levels of JAK2, STAT1, IRF-1, and PD-L1 were determined by qRT-PCR. (C, D) After treatments with AZD1480 or fludarabine under the same conditions as (B), the protein levels of STAT1, IRF-1, and PD-L1 proteins were determined by immunoblotting and quantified relative to β-actin. The data are presented as mean ± SEM (n = 3). *P < 0.05 and **P < 0.001 compared to only IFNγ-stimulated SNU-719 cell line (Student’s t-test).