Reconstitution of functional STAT1 in the AGS-1 cell line. ( a ) Immunoblot analysis of lysates from AGS-1 STAT1 cells using an anti-STAT1 antibody indicated that expression of STAT1 protein was restored. Ctrl, control. ( b ) The ability of STAT1 protein from AGS-1 STAT1 cells to undergo phosphorylation at Tyr-701 was confirmed by immunoblotting with an anti-P-STAT1 monoclonal antibody. ( c ) Overnight stimulation with IFN-α (10 3 U/ml) resulted in enhanced H2k expression on AGS-1 STAT1 but not AGS-1 MSCV melanoma cells. Shaded histograms represent fluorescence intensity of H2k-PE staining in IFN-α–stimulated EGFP-positive cells. White histograms represent H2k-PE staining in PBS-stimulated EGFP-positive cells. Determination of H2k-positive cells was based on isotype control antibodies (M1), which fell within the first log of fluorescence. ( d ) IFN-α inhibits proliferation of the AGS-1 STAT1 cell line in a dose-dependent manner. In contrast, the antiproliferative effects of IFN-α were not evident in AGS-1 MSCV cells. Results represent the mean of triplicate wells and are expressed as optical density at 570 nm ± SE. ( e ) The ability of the AGS-1 STAT1 cell line to regulate ISGF3 gene transcription in response to IFN-α was confirmed by PCR. Total cellular RNA was isolated from cell lines (B16F1, AGS-1, AGS-1 STAT1 , and AGS-1 MSCV ) after overnight treatment with 10 4 U/ml of murine IFN-α and converted to cDNA using standard methods. PCR primers specific for muISG-54 detected increased ISG-54 expression in only the B16F1 (positive control) and AGS-1 STAT1 cell lines after IFN-α treatment. Primers specific for 28s rRNA (housekeeping gene) were used as controls.
{}