Figure 1.
Prominent nuclear localization of STAT3-GFP independent of tyrosine phosphorylation.
A) Western blot of cell lysates demonstrate tyrosine phosphorylation of STAT3 and STAT3-GFP in response to cytokine. 293 cells were transfected with untagged STAT3 or STAT3-GFP expression plasmids, serum-starved, and either untreated or treated with interferon-α (IFNα) for one hour. Separate blots using antibodies to STAT3 or to the specific phosphorylated tyrosine 705 of STAT3 (p-Y-STAT) are shown B) Induction of a GAS-luciferase reporter gene by STAT3 or STAT3-GFP. Cells were transfected with the reporter gene and genes encoding GFP, STAT3, or STAT3-GFP, and were untreated (open bar) or treated with IFNα (black bar). Fold induction is shown relative to Renilla luciferase transfection controls. C) Live cell images of Hep3B cell co-expressing DsRed and STAT3-GFP after serum starvation (time 0) or stimulation with IL-6 (20 ng/ml) for 60 minutes. Nuclear fluorescence (FL) intensity was quantified with time for both fluorescent molecules in the same cell (graph). Results are representative of multiple independent experiments.
Figure 2.
Time-lapse imaging with photoactivation or photobleaching reveals STAT3-GFP continuous nuclear import.
A) Nuclear import of photoactivatable STAT3 (STAT3-PA-GFP). HeLa cells expressing STAT3-PA-GFP were serum-starved. A region in the cytoplasm (solid dot) was subjected to continuous high-intensity laser to photoactivate the cytoplasmic STAT3-PA-GFP and its nuclear accumulation was evaluated. B) Nuclear FRAP experiments were performed with cells expressing STAT3-GFP and serum-starved. A ROI in the nucleus was subjected to high intensity laser (black circle) to bleach nuclear STAT3-GFP. Nuclear fluorescence recovery was evaluated in untreated cells (control) or cells treated with IFNα. Fluorescence intensity was quantified in a ROI in cytoplasm (C) and the nucleus (N) in both untreated (control) and cytokine-treated cells and is shown graphically below. C) Multiple experiments with nuclear FRAP of STAT3-GFP were evaluated in HeLa cells treated with IFNα or Hep3B cells treated with IL-6. The half-time (T1/2) of nuclear fluorescence recovery was calculated by curve-fitting analysis (GraphPad Prism software) to evaluate STAT3-GFP nuclear import rates in untreated (control) (open bar) or cytokine-treated (black bar) cells.
Figure 3.
STAT3-GFP nuclear export independent of cytokine stimulation.
A) Photoactivation of nuclear STAT3-PA-GFP in HeLa cells. A ROI in the nucleus (solid dot) of serum-starved cells was subjected to high intensity laser activation with 2-photon laser microscopy. B) Cytosolic FLIP assays were performed in serum-starved HeLa cells expressing STAT3-GFP either untreated (control) or treated with IFNα. A ROI in the cytoplasm (black circle) was subjected to continuous laser bleaching. Time-lapse imaging was used to evaluate loss of fluorescence in the nucleus. Quantitation of fluorescence loss in the nucleus in untreated (control) or IFNα treated cells is shown graphically below. C) Kinetics of STAT3-GFP nuclear loss of fluorescence was quantified by curve-fitting analyses of multiple experiments. Results are shown for HeLa cells untreated (control) or treated with IFNα and Hep3B cells untreated or treated with IL-6.
Figure 4.
STAT3-STAT3 protein interaction in unphosphorylated and tyrosine phosphorylated states analyzed by FRET.
A) HeLa cells co-expressing STAT3-YFP and STAT3-CFP were serum starved, fixed, and analyzed for STAT3-STAT3 interactions by FRET. A ROI in the nucleus (black circle) was subjected to laser for gradual photobleaching of STAT3-YFP. Quantification of photobleaching the STAT3-YFP acceptor and the concomitant change in STAT3-CFP fluorescence (FRET) are shown graphically in the right panel. B) FRET efficiency was measured between STAT3-YFP and STAT3-CFP in the nucleus (N) or the cytoplasm (C) of multiple serum-starved cells untreated (control) or IFNα-treated cells. C) Amino terminal domain of STAT3 is required for unphosphorylated protein interaction. FRET efficiency was measured between STAT3(135–770)-YFP and STAT3(135–770)-CFP in the nucleus or the cytoplasm of multiple untreated (control) cells or IFNα-treated cells.
Figure 5.
STAT3-GFP is excluded from mitochondria.
A) HeLa cells expressing STAT3-GFP were stained with MitoTracker Orange and the localization of STAT3-GFP and mitochondria was captured with live cell imaging. B) HeLa cells co-expressing STAT3-GFP and YFP-RasV12 were stained with MitoTracker Orange and live cell imaging identified the localization of YFP-RasV12, STAT3-GFP, and mitochondria. C) HeLa cells expressing MLS-GFP were stained with MitoTracker Orange and live cell imaging captured the localization of MLS-GFP and mitochondria. Images captured with Zeiss LSM 5 using maximal vertical resolution (<1 µm) and either 40× oil objective or 63× C-Apochromat (water) objective.
Figure 6.
STAT3 nuclear import is dependent on Ran.
A) Nuclear FRAP experiments were performed to photobleach STAT3-YFP in the nucleus of Hep3B cells co-expressing either CFP-Ran wild type (CFP image top left panel) or CFP-Ran Q69L (CFP image bottom left panel). A ROI in the nucleus was subjected to high intensity laser (black circle) to bleach nuclear STAT3-YFP. YFP fluorescence recovery in the nucleus was followed with time-lapse imaging in cells expressing CFP-Ran wt (top panels) or CFP-Ran Q69L (bottom panels). Quantitation of STAT3-YFP in the nucleus in shown graphically below the microscopic images. Fluorescence was measured in a ROI in the nucleus (N) and a ROI in the cytoplasm (C) in cells expressing wt Ran or Ran Q69L. B) Multiple HeLa or Hep3B cells co-expressing STAT3-YFP with either wt Ran or Ran Q69L were evaluated prior to photo-bleaching. The percentage of cells expressing greater nuclear than cytoplasmic fluorescence (N>C) or greater cytoplasmic to nuclear fluorescence (C>N) of STAT3-YFP was measured.
Figure 7.
Knockdown of importin-β1 inhibits STAT3-GFP nuclear import.
HeLa cells were transfected with vimentin siRNA (control) or importin-β1 siRNA and STAT3-GFP. A) Images of STAT3-GFP localization. Four independent experiments indicated 100% of cells expressing control siRNA had prominent nuclear STAT3-GFP. In cultures expressing importin-β1 siRNA, an average of 58% of cells showed a defect in STAT3-GFP nuclear import, with 18% of cells showing the severe impairment indicated in the image. B) Effective knockdown of importin-β1 mRNA (Imp-β1) relative to GAPDH mRNA measured in cells expressing importin-β1 siRNA or vimentin siRNA (CTRL) by RT-PCR. Average knockdown of importin-β1 mRNA from four experiments was approximately 56% measured with Image J software. C) Knockdown of importin-β1 protein relative to tubulin was approximately 50% evaluated by Western blot.