American Society of Hematology

Figure 1

$Figure 1. SUMO-mediated paracrystal dispersal curtails STAT1 activity in the nucleus. (A) Green fluorescent protein (GFP)–tagged wild-type STAT1 or mutant Glu705Gln (ΔSUMO) was coexpressed with Ubc9 (all lanes) and His-tagged SUMO1 (lanes 1, 2, 4, and 5) in HEK293T cells. Whole cell extracts were prepared in buffer containing 120mM N-ethyl-maleimide (Sigma-Aldrich) and subjected to native affinity chromatography on Ni-NTA agarose (QIAGEN) to enrich His-tagged SUMO1 conjugates. Shown are the results of a representative immunoblot analysis of cell extracts and bound Ni-NTA chromatography fraction using anti STAT1-specific antibody (C24; Santa Cruz Biotechnology). (B) Left: Immunofluorescence confocal micrographs of unstimulated or 1-hour IFNγ-stimulated (50 U/mL mouse IFNγ; Calbiochem) mouse embryonic fibroblasts derived from SUMO-free STAT1-Glu705Gln (ΔSUMO) knockin mice or wild-type littermates using anti–Tyr701-phosphorylated STAT1 antibody. Nuclei were stained with Hoechst dye. Right: immunoblot analyses of corresponding whole cell extracts depicting STAT1 activation kinetics. Cells were left untreated or were treated with IFNγ for 60 minutes, after which time the medium was replaced by growth medium without or with 0.5μM tyrosine kinase inhibitor staurosporine. The cells were incubated for the indicated times before cell extraction and consecutive Western blotting on the same membrane using anti-Tyr701–phosphorylated STAT1-specific antibody (Cell Signaling Technology), anti-STAT1–specific antibody (E23; Santa Cruz Biotechnology) and then anti–β-actin specific antibody (Sigma-Aldrich). Note that antibody E23 recognizes both STAT1 splice variants, full-length STAT1α, and truncated STAT1β. (C) Diagram depicting specific Tyr701-phosphorylation of SUMO-free and wild-type STAT1 using fibroblast extracts as shown in panel B. IFNγ-stimulated wild type was set as 100. Data are presented as the mean ± SD of 3 independent immunoblot analyses for each STAT1 variant. (D) Left: Immunofluorescence confocal micrographs of unstimulated or 1-hour IFNγ-stimulated (50 U/mL mouse IFNγ) BMMs from SUMO-free STAT1 (ΔSUMO) knockin mice or wild-type littermates using anti Tyr701-phosphorylated STAT1-specific antibody. Cell dimensions are shown using bright-field microscopy. Right: Representative immunoblot analyses of corresponding whole cell extracts depicting STAT1 phosphorylation kinetics at residues Ser727 and Tyr701. Anti-Ser727–phosphorylated STAT1-specific antibody (44-382G; Invitrogen), anti-Tyr701–phosphorylated STAT1-specific antibody (Cell Signaling Technology), and then anti-STAT1–specific antibody (M23; Santa Cruz Biotechnology; note that this antibody recognizes full-length STAT1α splice variant only) were used. (E) Time course of soluble activated STAT1 in the nucleus of HEK293T cells expressing wild-type or SUMO-free STAT1, as determined by quantitative immunofluorescence confocal microscopy. Top left: Representative immunofluorescence micrographs of cells before and after treatment with 5 ng/mL human IFNγ (Calbiochem) using anti-Tyr701–phosphorylated STAT1-specific antibody. Bottom: Scatter plot depicting unprocessed fluorescence signal intensities recorded outside paracrystals and nucleoli in the nucleoplasm of 170-250 randomly selected cells per time point for each STAT1 variant. For SUMO-free STAT1, cells with paracrystals and without are grouped separately; horizontal bars indicate average fluorescence signal intensity. Top right: Graph depicting the time course of average fluorescence signal intensities. Data were background subtracted; in addition, to correct for the ∼ 15% of cells that were unresponsive to IFNγ (not applicable to dataset ΔSUMO with paracrystals), the bottom 15% intensities of each time point were excluded in this representation. Data are shown relative to the wild-type maximum (t = 2 h), which was set as 100.$

SUMO-mediated paracrystal dispersal curtails STAT1 activity in the nucleus. (A) Green fluorescent protein (GFP)–tagged wild-type STAT1 or mutant Glu705Gln (ΔSUMO) was coexpressed with Ubc9 (all lanes) and His-tagged SUMO1 (lanes 1, 2, 4, and 5) in HEK293T cells. Whole cell extracts were prepared in buffer containing 120mM N-ethyl-maleimide (Sigma-Aldrich) and subjected to native affinity chromatography on Ni-NTA agarose (QIAGEN) to enrich His-tagged SUMO1 conjugates. Shown are the results of a representative immunoblot analysis of cell extracts and bound Ni-NTA chromatography fraction using anti STAT1-specific antibody (C24; Santa Cruz Biotechnology). (B) Left: Immunofluorescence confocal micrographs of unstimulated or 1-hour IFNγ-stimulated (50 U/mL mouse IFNγ; Calbiochem) mouse embryonic fibroblasts derived from SUMO-free STAT1-Glu705Gln (ΔSUMO) knockin mice or wild-type littermates using anti–Tyr701-phosphorylated STAT1 antibody. Nuclei were stained with Hoechst dye. Right: immunoblot analyses of corresponding whole cell extracts depicting STAT1 activation kinetics. Cells were left untreated or were treated with IFNγ for 60 minutes, after which time the medium was replaced by growth medium without or with 0.5μM tyrosine kinase inhibitor staurosporine. The cells were incubated for the indicated times before cell extraction and consecutive Western blotting on the same membrane using anti-Tyr701–phosphorylated STAT1-specific antibody (Cell Signaling Technology), anti-STAT1–specific antibody (E23; Santa Cruz Biotechnology) and then anti–β-actin specific antibody (Sigma-Aldrich). Note that antibody E23 recognizes both STAT1 splice variants, full-length STAT1α, and truncated STAT1β. (C) Diagram depicting specific Tyr701-phosphorylation of SUMO-free and wild-type STAT1 using fibroblast extracts as shown in panel B. IFNγ-stimulated wild type was set as 100. Data are presented as the mean ± SD of 3 independent immunoblot analyses for each STAT1 variant. (D) Left: Immunofluorescence confocal micrographs of unstimulated or 1-hour IFNγ-stimulated (50 U/mL mouse IFNγ) BMMs from SUMO-free STAT1 (ΔSUMO) knockin mice or wild-type littermates using anti Tyr701-phosphorylated STAT1-specific antibody. Cell dimensions are shown using bright-field microscopy. Right: Representative immunoblot analyses of corresponding whole cell extracts depicting STAT1 phosphorylation kinetics at residues Ser727 and Tyr701. Anti-Ser727–phosphorylated STAT1-specific antibody (44-382G; Invitrogen), anti-Tyr701–phosphorylated STAT1-specific antibody (Cell Signaling Technology), and then anti-STAT1–specific antibody (M23; Santa Cruz Biotechnology; note that this antibody recognizes full-length STAT1α splice variant only) were used. (E) Time course of soluble activated STAT1 in the nucleus of HEK293T cells expressing wild-type or SUMO-free STAT1, as determined by quantitative immunofluorescence confocal microscopy. Top left: Representative immunofluorescence micrographs of cells before and after treatment with 5 ng/mL human IFNγ (Calbiochem) using anti-Tyr701–phosphorylated STAT1-specific antibody. Bottom: Scatter plot depicting unprocessed fluorescence signal intensities recorded outside paracrystals and nucleoli in the nucleoplasm of 170-250 randomly selected cells per time point for each STAT1 variant. For SUMO-free STAT1, cells with paracrystals and without are grouped separately; horizontal bars indicate average fluorescence signal intensity. Top right: Graph depicting the time course of average fluorescence signal intensities. Data were background subtracted; in addition, to correct for the ∼ 15% of cells that were unresponsive to IFNγ (not applicable to dataset ΔSUMO with paracrystals), the bottom 15% intensities of each time point were excluded in this representation. Data are shown relative to the wild-type maximum (t = 2 h), which was set as 100.

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