Replacement of glutamine residues in position 559 and 563 results in STAT1 mutants with a prolonged IFNγ-induced nuclear accumulation.

<p>(A, B) HeLa cells expressing GFP fusion proteins with either STAT1-WT, -E559A, -E563A, or -K567A were untreated or stimulated for 45 min with 5 ng/ml human IFNγ followed by exposure to staurosporine (500 nM) for 0 min, 60 min and 120 min, respectively. The fluorescence micrographs show the intracellular distribution of the GFP-tagged fusion proteins and the localization of the corresponding Hoechst-stained nuclei (scale bar 10 µm). (B) Quantification of the nucleocytoplasmic STAT1-GFP distribution in untreated and IFNγ-pretreated cells exposed to staurosporine as determined from experiment (A). The histograms demonstrate the ratio of nuclear-to-total cellular fluorescence intensities as measured for the indicated STAT1 variants and stimulation modes. (C) Indirect fluorescence microscopy confirming the decreased nuclear export rates of STAT1-E559A and -E563A. STAT1-negative U3A cells, reconstituted with untagged wild-type or mutant STAT1, were treated as above, except that exposure times to staurosporine were shortened (0 min, 30 min, and 60 min). Intracellular distribution of recombinant STAT1 in the fixed, Hoechst-stained cells was monitored immunocytochemically using anti-STAT1 C-24 and Cy3-labelled secondary antibodies (scale bar 10 µm). (D) Nucleocytoplasmic distribution of the STAT1 mutants, as quantified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097633#pone-0097633-g002" target="_blank">Figure 2C</a>, with bars and asterisks indicating significant differences between wild-type and the respective mutant.