Fig. 3

Unaltered DNA binding, reporter activation and target gene induction by WT STAT1 in the presence of a dimerization-deficient mutant. A Electrophoretic mobility shift assay demonstrated unchanged binding of the recombinant WT protein from reconstituted U3A cell extracts to a [³³P]-radioactively labelled M67 probe containing a single GAS sequence, whereas the co-expressed unphosphorylated STAT1 variants and empty vector (pEGFPN1) showed no DNA-binding indicated by the absence of a GFP-tagged STAT1 band in lanes 3 to 5. The asterisk at the margin of the gelshift indicates an unspecific band. B EMSA result showing the unaltered capacity of endogenous STAT1 from HeLa cell extracts to form tetramers on the radioactively-labelled DNA probe 2xGAS, containing two GAS sites in tandem orientation, in the presence of co-expressed QM-GFP. HeLa cells were either untransfected (UT) or transfected with the indicated expression plasmids, and on the next day the cells were treated for 45 min with IFNγ or left untreated. Extracts were incubated with 2xGAS DNA probed for 15 min before subsequently being challenged by a 750-fold molar excess of unlabelled M67 DNA incubated for 10 min at RT in a competition assay (comp). For super-shift assays, 20 ng of STAT1- (αS1) or STAT3-specific (αS3) antibody were added to the reaction for 40 min at RT. C, D EMSA result showing the unaltered cytoplasmic and nuclear fractions of endogenous P-STAT1 from HeLa cell lysates bound to a [³³P]-radioactively labelled M67 probe containing a single GAS sequence, as the additional presence of QM-GFP did not hamper nuclear import of the endogenous STAT1 but its nuclear retention. E HeLa cells expressing WT-GFP, empty vector (pEGFPN1) or the quadruple mutant QM-GFP were left untreated (−) or stimulated for 6 h with 50 ng/ml of IFNγ (+). In whole extracts from these cells, luciferase luminescence of a reporter construct with a triple GAS site (3xLy6E) and the enzymatic activity of the co-expressed β-galactosidase were measured and represented graphically. F U3A cells were transfected with a STAT1-responsive luciferase reporter construct, a β-galactosidase expression vector and a combination of equal amounts of WT-GFP and the indicated STAT1 variants. These cells were either unstimulated (−) or stimulated with 50 ng/ml of IFNγ (+) and subsequently luciferase activity, normalized to the β-galactosidase expression, was measured in whole cell extracts and represented graphically. The experiment was repeated in six independent transfections at least three times. G–J U3A cells were untransfected (UT) or transfected with a plasmid coding for WT-GFP alone or a combination of WT-GFP and the indicated U-STAT1 variants or empty GFP vector (pEGFPN1). These cells where either untreated or stimulated with 50 ng/ml of IFNγ for 6 h. after which RNA was isolated and converted to cDNA. Histograms show the results from qPCR experiments for the following STAT1 target genes: G GBP1, H MIG, I CXCL10 and J IRF1. Gene induction was normalized to the expression of the house-keeping gene GAPDH. Histograms show means and standard deviations wherein the significant differences between the IFNγ-stimulated variant samples and cells expressing a single transfection of WT protein are marked by bars and asterisks. The experiment was repeated three times