Figure 1.

Characterization of the AGS-1 cell line. AGS-1 has the appearance of a malignant melanoma by (a) hematoxylin and eosin staining and immunohistochemistry for (b) S-100 and (c) HMB-45. (d) Immunoblot analysis confirmed that the AGS-1 cell line was STAT1 deficient (lane 3). Cell lysates from the A431 cell line (lane 1) and B16F1 cell line (lane 2) were used as positive controls. The protein content of the lysates was quantitated, and equal amounts were loaded onto the gel. (e) EMSA analysis revealed no activation of STAT1 in IFN-α–treated AGS-1 cells. Lysates from IFN-α–stimulated B16F1 cells were used as positive controls in this assay. SIS, SIS-inducible element. (f) Flow cytometric analysis of H2k expression. Treatment of B16F1 cells for 48 hours with 10⁴ U/ml muIFN-α resulted in increased H2k expression. Loss of STAT1 significantly reduced the induction of H2k in the AGS-1 cell line after IFN-α treatment. The minor induction of H2k expression by IFN-α in the AGS-1 cell line can be attributed to STAT1-independent signaling through NF-κB (28, 29). (g) Northern blot analysis indicated a loss of ISG-54 gene regulation in AGS-1 cells after stimulation with 10⁴ U/ml muIFN-α for 16 hours. Probes specific for GAPDH (housekeeping gene) were used as controls and did not vary across samples. (h) Northern blot findings were confirmed by PCR using primers specific for muISG-54 and showed increased ISG-54 expression only in B16F1 cells (positive control) after IFN-α treatment. Primers specific for 28s rRNA (housekeeping gene) were used as a controls in this assay. Neg, negative control.