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First published online 2 December 2003
doi: 10.1242/jcs.00833

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STAT3 is enriched in nuclear bodies

¹ Institut für Biochemie,, Universitätsklinikum RWTH Aachen, Pauwelsstraße 30, 52057 Aachen, Germany
² Institut für Pharmakologie und Toxikologie, Universitätsklinikum RWTH Aachen, Pauwelsstraße 30, 52057 Aachen, Germany

View larger version (42K): [in a new window]   Fig. 1. Biochemical and functional characterization of STAT3-YFP. (A) Domain structures of STAT3 and STAT3-YFP. Domain borders were chosen according to Becker et al. (Becker et al., 1998). (B) The fusion protein STAT3-YFP is a functional player. COS-7 cells were cotransfected with expression vectors encoding STAT3 or STAT3-YFP and chimeric IL-5R/gp130 receptor chains as indicated. Cells were stimulated with IL-5 (20 ng/ml) for 30 minutes or left unstimulated. Lysates were analyzed by western blotting (WB) using a STAT3 phosphotyrosine-specific antibody (upper panel). After stripping, the blot was reprobed with a STAT3 antibody (lower panel). (C) Cells transfected as described above were stimulated with IL-5 (20 ng/ml) for various time periods as indicated. STAT3 DNA-binding activity was monitored by an EMSA using the sis-inducible element (SIE)-probe. Supershifts were performed using STAT3 (*) or GFP (#) antibodies. (D) HepG2 cells were cotransfected with a STAT3-specific luciferase-reporter gene plasmid (m67-SIE-TK-luc), a plasmid encoding ß-gal for determination of transfection efficiency, and mock vector, STAT3, or STAT3-YFP as indicated. Cells were stimulated for 16 hours with 20 ng/ml IL-6 (dark gray bars) or left untreated (light gray bars). Relative luciferase activities were normalized with ß-gal activities. Mean values of experiments performed in triplicate are depicted.

View larger version (55K): [in a new window]   Fig. 2. STAT3 in nuclear bodies. (A) Untransfected HepG2 cells (upper panels) or HepG2 cells transfected with STAT3 (lower panels) were stimulated with 20 ng/ml IL-6 for 15 minutes or left unstimulated as indicated. Cells were fixed and STAT3 was immunostained with a STAT3-specific antibody and a TRITC-conjugated secondary antibody. Images were taken by confocal microscopy. Endogenous (upper panels) as well as transfected (lower panels) STAT3 was detected. Bars, 10 µm. (B) STAT3-YFP nuclear translocation analyzed by live cell imaging. HepG2 cells transfected with STAT3-YFP were placed in a perfusion chamber at 37°C and analyzed by confocal laser scanning microscopy. Cells were stimulated with 20 ng/ml IL-6 and pictures of a single cell were taken every 30 seconds. Images taken at the timepoints indicated are depicted. Bar, 10 µm. A time-lapse movie can be downloaded as supplementary data on the homepage of the Journal of Cell Science (jcs.biologists.org). (C) Kinetics of STAT3 and STAT3-YFP phosphorylation. HepG2 cells were transfected with mock vector or STAT3-YFP. Lysates were prepared after stimulation with 20 ng/ml IL-6 for the timepoints indicated. Endogeneous STAT3 (endo) and STAT3-YFP were precipitated by a STAT3-specific antibody (upper panels). STAT3-YFP was precipitated by a GFP-specific antibody (lower panels). Tyrosine phosphorylation of STAT3 and STAT3-YFP was detected by western blot analysis using a STAT3 phosphotyrosine-specific antibody. After stripping the blots were reprobed with a STAT3 antibody for loading control.

View larger version (93K): [in a new window]   Fig. 3. Phosphorylation of STAT3 in nuclear bodies. HepG2 cells transiently transfected with STAT3-YFP were stimulated with IL-6 (20 ng/ml) for 30 minutes or left unstimulated as indicated. Subsequently, cells were fixed and incubated with (A) STAT3 phosphotyrosine-specific antibodies or (B) STAT3 phosphoserine-specific antibodies. A TRITC-conjugated secondary antibody was used for immunostaining. Cells were analyzed by confocal microscopy. Bars, 10 µm.

View larger version (47K): [in a new window]   Fig. 4. FRAP analysis of STAT3-YFP mobility in the nucleus. HepG2 cells were transfected with STAT3-YFP and living cells were stimulated with 20 ng/ml IL-6. (A) 15 minutes after stimulation, a single STAT3-YFP nuclear body was bleached using the 514 nm laser of the laser-scanning microscope. Subsequently, images were taken at the timepoints indicated. Bar, 10 µm. (B) For quantitative FRAP analysis, ROIs of 1 µm in diameter within the nuclei of STAT3-YFP-transfected HepG2 cells before (blue graph) and 15 minutes after IL-6 stimulation (green graph) were bleached and subsequently the recovery of fluorescence was monitored. Fluorescence before bleaching was normalized to 100. YFP was transfected into HepG2 cells as a control for a freely mobile protein. Unstimulated cells were analyzed as described above (black graph). Each graph represents the mean values of five independent experiments.

View larger version (73K): [in a new window]   Fig. 5. Localization of SC-35, GFP-SF2/ASF, PML and STAT3. (A) HepG2 cells were transfected with STAT3-YFP. Cells were stimulated with IL-6 for 30 minutes or left unstimulated as indictated. Subsequently, cells were fixed and SC-35 was immunostained with a SC-35-specific antibody and a TRITC-conjugated secondary antibody. Images were taken by confocal microscopy. Bars, 10 µm. (B) HepG2 cells were cotransfected with the splicing factor fusion protein GFP-SF2/ASF and STAT3. After IL-6 treatment for 15 minutes cells were fixed and STAT3 was immunostained with a STAT3-specific antibody and a TRITC-conjugated secondary antibody. Images were taken by confocal microscopy. Bars, 10 µm. (C) HepG2 cells were transfected with STAT3-YFP. Cells were stimulated with IL-6 for 30 minutes or left unstimulated. Subsequently, cells were fixed and PML was immunostained with a PML-specific antibody and a TRITC-conjugated secondary antibody. Images were taken by confocal microscopy. Bars, 10 µm.

View larger version (94K): [in a new window]   Fig. 6. Localization of acetylated histone H4, CBP and STAT3. HepG2 cells were transfected with STAT3-YFP. Cells were stimulated with 20 ng/ml IL-6 for 30 minutes or left unstimulated. Subsequently, cells were fixed and (A) CBP or (B) acetylated histone H4 was immunostained using the respective specific antibodies and a TRITC-conjugated secondary antibody. Images were taken by confocal microscopy. White circles mark selected STAT3 bodies that colocalize with domains of CBP or acetylated histone H4, respectively. Bars, 10 µm.

View larger version (66K): [in a new window]   Fig. 7. Localization of nascent RNA and STAT3 nuclear bodies. HepG2 cells were transfected with STAT3-YFP. Fortyeight hours after transfection cells were pre-incubated in medium supplemented with 1 mM bromouridine for 30 minutes. Subsequently, cells were stimulated with 20 ng/ml IL-6 for another 30 minutes in the presence of bromouridine. Images of fixed cells were taken by confocal laser-scanning microscopy (upper panel). Intensities of the BrU and STAT3-YFP signals are also shown in false-color mode, indicating highest intensities in white and red and lowest intensities in blue and black (lower panel). White circles mark a selected area where high levels of STAT3 and nascent RNA were detected. Bars, 10 µm.

View larger version (70K): [in a new window]   Fig. 8. Actinomycin D has no effect on STAT3-YFP nuclear body formation. HepG2 cells were transfected with STAT3-YFP, preincubated with 4 µM actinomycin D in DMSO for 2 hours or left without inhibitor and were subsequently treated for 30 minutes with 20 ng/ml IL-6 or left untreated as indicated in the figure. Images of fixed cells were taken by confocal laser-scanning microscopy. DMSO treatment alone was taken as a control. Bars, 10 µm.

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