Fig. 6. Nuclear translocation of STAT1 and IFNGR1 by intracellular IFN requires the NLS of IFN. (A) WISH cells were transfected for 8 hours with an empty vector (column 1), a vector expressing non-secreted IFN (column 2) or a vector expressing NLS-modified IFN (column 3) and stained simultaneously with antibodies to STAT1 and IFNGR1. Secondary antibodies to STAT1 conjugated to Alexa 594 (top row) or to IFNGR1 conjugated to Cy-2 (bottom row) were used and analyzed by fluorescence microscopy. (B) Quantitation of images. Images of cells transduced with an empty vector control (left lanes), a vector expressing non-secreted IFN (middle lanes) or a vector expressing NLS-mutated IFN (right lanes) were viewed in seven different fields to a obtain mean ratio of nuclear pixel intensity (Fn) to cytoplasmic pixel intensity (Fc). STAT1 and IFNGR1 Fn/Fc for nsIFN versus mutant were both significant at P<0.002 by t-test. Calculations for fluorescence in the nucleus (N) versus cytoplasm (C) were also done by using N/N+C. Four independent measurements showed a P<0.03 by t-test for the nuclear translocation of STAT1 and IFNGR1 for the wild-type IFN versus the NLS-mutated IFN.