100, 1% Trasyslol (Bayer, Leverkusen, Germany), 10 mg/ml Leupeptin). Extracted cells were washed shortly with this buffer lacking the detergent and fixed with 1% paraformaldehyde in this buffer for 15 min. Intact cells were fixed in cold (220uC) acetone for 30 min. An alternative approach was to fix the cells with 1% paraformalde- hyde in phosphate buffered saline (PBS) for 20 min and to permeabilize subsequently with 0.2% Triton X-100 in this buffer. Residual free aldehyde groups were blocked with 50 mM glycine in PBS for 10 min. Acetone fixed cells were rehydrated in PBS for 10 min, blocked with 2% normal serum for 20 min, and labeled with the primary antibody. Immunogold labelling of cytoskeletons and handling of the probes for electron microscopy were done as described in detail elsewhere [37]. Goat anti-mouse- and goat anti- rabbit gold conjugates were purchased from British Biocell International, Cardiff, UK. Flow cytometry Cells grown on 100 mm Petri dishes were trypsinized, washed once with PBS, resuspended in 0.5 ml PBS and fixed in 70% cold methanol. After rehydration in PBS, the cells were suspended in 1 ml of propidium iodide solution (5 mg propidium iodide, 1 mg RNase A per ml of PBS) for 30 min at 37uC. Labeled cells were run through a Coulter Epics XL-MCL Counter (Beckman- Coulter, U.S.A.). The raw data were converted into single- parameter histograms using the WinMDI 2.8 software and saved as FCS files. The quantitative cell cycle analysis was performed with the Cylchred program (University of Cardiff, England). Image acquisition and image processing Images were taken with the confocal microscope Zeiss LSM 510 Meta (Zeiss, Jena, Germany). Image processing was done with Photoshop version 7.01, and with ImageJ 1.33 (NIH, public domain, U.S.A.). Image processing included the following steps:(i) correction of tonal value including adjustment of threshold and gamma value, (ii) noise suppression with low pass filters, (iii) sharpening through overlay of highpass (Laplace) and bandpass filtered original images. Merged images were generated by setting signals below the threshold value to 50% gray. Quantitative colocalization analysis, including the determination of Pearson’s correlation coefficient were done with raw confocal data. Image data were acquired at Nyquist sampling rates. Iterative deconvolu- tion was done, when necessary, with the Zeiss Advanced Imaging Microscopy (AIM) Software v. 3.2 in an automated modus. Colocalization analysis and scattergramms were calculated with the Zeiss Advanced Imaging Microscopy (AIM) Software v. 3.2 and JACoP [36] under ImageJ v. 1.41o. Immunoprecipitation and Western blotting Subconfluent cultures were washed twice with cold (4uC) PBS. The cells were scraped from the culture dish and lysed with 1 ml of Hepes-buffer pH 7.0 (150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 0.1% NP40; 1% aprotinin ( Bayer; Leverkusen, Germany); 10 mg/ml Leupeptin) while shaking on ice for 30 min. The lysates were cleared in an Eppendorf microfuge at 13,000 rpm for 30 min. The protein concentration in the supernatant lysate was determined colorimetrically with the ‘BCA Protein Assay’ reagent (Pierce, Rockford, USA). An aliquot containing 100 mg of protein was adjusted to a total volume of 300 ml with lysis buffer. The probes were incubated for 2 h at 4uC with 2 mg of the appropriate antibody prebound to 100 ml of a 10% slurry of protein G- Nestin and Steroid Receptor sepharose. The PGS with the bound immune complexes was washed three times with cold lysis buffer. Precipitated proteins were dissolved in 20 ml SDS sample buffer, electrophoresed on 8% respectively 10% polyacrylamide gels, and transferred onto nitrocellulose (Hybond, Amersham, England). The nitrocellulose sheets were stained with Amido black, destained, and blocked with 5% low fat milk in Tris-buffered salt solution (20 mM Tris/HCl, pH 7.6; 150 mM NaCl) containing 0.05% Tween-20. Proteins were detected with the appropriate primary antibody and a secondary, enzyme conjugated antibody, using the ECL Western- blot detection kit (Amersham, Braunschweig, Germany). Chemi- luminescence was visualized by exposing the membrane to X-ray films. Silencing of GR and nestin The lentiviral control vector DNA containing a scrambled sequence and the vector DNA for silencing human GR and human nestin were obtained from Sigma, Germany. Lentiviral particles were produced in 293T cells by cotransfection of 3 mg vector DNA, 3 mg packaging construct pCMV-dR8.91 [obtained from D. Trono, University of Geneva], and 0.5 mg pMD2a expression construct. Viral particles were harvested after 48 h and stored at 280uC. For silencing of GR, THP-1 cells were infected with 500 ng (p24) lentiviral vector and selected for stably transduced cells with puromycin. Supporting Information Figure S1 GR colocalizes with vimentin in cytoskeletons of C6D8 cells. (A–C) Confocal images of cytoskeletons labelled with antibodies to vimentin (Vim), glucocorticoid receptor (GR), actin and tubulin (Tub); intensities below threshold value were set to 50% gray. Colocalization analysis was performed with raw confocal data. (A1–A3) Double staining with antibodies to GR and vimentin; (B1–B2) triple staining with antibodies to GR, actin and vimentin; (C1–C3) triple staining with antibodies to GR, tubulin, and vimentin; scatter diagrams indicate coincident signals of GR and vimentin (inserts in A3, B1, and C2), but not of GR and actin (insert in B1) or GR and tubulin (insert in C1). Bar in B = 5 mm; bars in A1and C1 = 1 mm. Found at: doi:10.1371/journal.pone.0006084.s001 (5.50 MB TIF) Figure S2 GR remains associated with the vimentin network in cells treated with cytochalasin B. Confocal images of cells; cells were incubated with 5 mg/ml cytochalasin B for 20 min, lysed in situ with Triton X-100 and stained with antibodies to vimentin, actin and GR; bar = 10 mm. Found at: doi:10.1371/journal.pone.0006084.s002 (2.59 MB TIF) Figure S3 Cytoskeleton bound GR colocalizes with HSC70. Confocal image of an extracted cell double labelled with antibodies to GR and HSC70. Bar = 1 mm. Found at: doi:10.1371/journal.pone.0006084.s003 (2.37 MB TIF) Acknowledgments The authors are grateful to J. Yang for supplying us with the nestin expression vector. Author Contributions Conceived and designed the experiments: RR HH WB. Performed the experiments: RR HH BS CH HH. Analyzed the data: WD. Contributed reagents/materials/analysis tools: CH HH WD. Wrote the paper: WB. PLoS ONE | www.plosone.org 14 June 2009 | Volume 4 | Issue 6 | e6084