PML Protein Expression is Inversely Correlated with the Level of IP-10.

(A) Immunohistochemical staining for PML and IP-10 protein expression was performed for 49 cases of stage IV advanced gastric carcinomas. Immunopositivity for PML protein was categorized as diffuse positivity (DP; nuclear immunoreactivity in ≥50% of tumor cells), focal positivity (FP; in ≥10% but <50%), or complete loss (CL; in <10%). For quantitative analysis of immunoreactivity of IP-10, positively stained areas were measured using an ImageJ software program as described in Materials and Methods. Representative images show PML and IP-10 protein expression in advanced gastric carcinoma tissues (right). (B) SNU-638 cells were transiently transfected with Pml siRNA or control siRNA. Two days after transfection, cells were incubated in the absence or presence of IFN-γ (10 ng/ml) for 8 h. IP-10 protein from the culture supernatants was analyzed by ELISA and normalized by the cell protein concentration. The relative concentration was calculated as the normalized amount divided by the normalized amount of SNU-638 cells which were transiently transfected with control siRNA in the presence of IFN-γ, and the concentration from parental cells was arbitrarily set at 100%. (C) SNU-638 gastric cancer cells were transiently transfected with either empty vector or PML IV expression vector. Day after transfection, cells were treated with or without IFN-γ (10 ng/ml) for 8 h and culture supernatants were obtained and subjected to ELISA as described in B. PML protein expression was verified for each assay by immunoblotting. Data shown are representative of at least three experiments. Bars, SD. *P<0.05, **P<0.01.