The HBZ-SP1 isoform of human T-cell leukemia virus type I represses JunB activity by sequestration into nuclear bodies.

Plasmids

The vectors pcDNA-HBZ-SP1-Myc, pEGFP-HBZ-bZIP (Fig. 1A ), pcDNA-JunB, pCMV-JunD-Flag have already been described [ 23 , 25 , 30 ]. To generate the EGFP fusion proteins, HBZ-SP1, HBZ-SP1ΔZIP, HBZΔAD, and HBZΔADΔbZIP DNA (Fig. 1A ) were PCR amplified from the pcDNA-HBZ-SP1-Myc vector, digested with Eco RI and Bam HI, and subcloned in frame into similarly digested pEGFP-C2 (Clontech). pEGFP-HBZΔAD and pEGFP-HBZΔADΔbZIP encode for the HBZ-SP1 protein from residues 77 to 206 and 77 to 132, respectively. pLCR-Luc and pNLS-EGFP (corresponding to EGFP under the control of the nuclear localization signal of SV40) is a gift from G. Steger and M. Piechaczyk, respectively.

Fluorescence microscopy analysis

COS cells were cultured in DMEM supplemented with 10% FCS. 24 h before transfection, cells were seeded onto glass slides. They were transfected using the jetPEI™ transfection reagent (Qbiogene) according to the manufacturer's instructions and, after 48 h, they were washed with PBS, fixed, and permeabilized with 4% paraformaldehyde and 0.1% Triton ×-100 for 30 min at room temperature. If necessary, cells were incubated with primary antibody (mouse anti-Myc antibody 9E10 or anti-Flag, Sigma-Aldrich; mouse anti-nucleolin, anti-JunB, or anti-RNA polymerase II antibody, Santa Cruz Biotechnology Inc.; the mouse anti-SC35 antibody is a gift from J. Tazi) for 1 h at room temperature. Samples were washed with PBS and then incubated with secondary FITC- or Texas Red-labelled antibodies (Pierce) for 1 h at room temperature. Coverslips were mounted with Vectashield containing DAPI (Abcys) for direct observation. Fluorescence images were acquired by fluorescence microscopy (model DM R; Leica) at room temperature with a 63x, NA 1.32, oil immersion objective at pinhole size 1 Airy (observation with immersion oil type DF, Cargille Laboratories Inc.). DAPI, GFP, FITC, and Texas Red were excited by 365-, 488-, 492-, and 596-nm laser light and emission was detected at 420, 507, 520, and 620 nm, respectively. The photographs were taken with a Photo Leika DC 250 camera and the images were analyzed with QFluoro software (Leica).

FRAP

FRAP was performed using the Zeiss LSM Meta 510 confocal microscope. FRAP recoveries were acquired at 37°C on EGFP-HBZ-SP1- or EGFP-HBZ-SP1ΔZIP-expressing cells plated on glass coverslips. The 488-nm line of the Ar+ laser was used for the excitation of EGFP. Cells were observed using a 63x, NA 1.2, oil immersion objective at pinhole size 1 Airy (observation with immersion oil Immersol™, Zeiss). After 5 prebleach scans, a region of interest was bleached and fluorescence recovery was analyzed for 5 min. Experimental recoveries were normalized and corrected for z-position fluctuation of cells using another region as an internal standard.

HeLa cell transfection and luciferase assay

HeLa cells were cultured in DMEM supplemented with 10% FCS and were transfected using the jetPEI™ transfection reagent (Qbiogene). 1 μg of pcDNA3.1(-)/Myc-His/ lacZ (β-galactosidase-containing reference plasmid) was included in each transfection to control for transfection efficiency. The total amount of DNA in each transfection was the same (3.6 μg) through the adequate added amount of pcDNA3.1(-)/Myc-His. Cell extracts normalized for protein content were used for luciferase and β-galactosidase assays as already described [ 30 ].

Cell cycle analysis by flow cytometry

Cell cycle analysis was based on the measurement of nuclear DNA content using flow cytometry and propidium iodide. Briefly, HeLa cells were trypsinized, washed with PBS, and fixed in cold 70% ethanol. After being washed in PBS, cells were resuspended in PBS containing 0.1% Triton ×-100, 40 μg/ml propidium iodide, and 50 μg/ml RNase A. Stained cells were then processed with an EPICS XL4C cytofluorometer (Coulter) and analyzed with the CellCycle software. For flow cytometric analysis of transiently transfected cells, 10 6 HeLa cells were transfected with 5 μg of pNLS-EGFP or pEGFP-HBZ-SP1 using the BD Calphos™ mammalian transfection kit (BD Biosciences) according to the manufacturer's instructions. At 24 h posttransfection, cells were harvested and GFP-positive cells were then analyzed for DNA content with a UV laser by the EPICS XL4C cytofluorometer (Coulter). For analyses of S-phase entry, the cells were arrested by using a double thymidine block. The HeLa cells were blocked for 12h with 2,5 mM thymidine, washed with DMEM without serum, transfected with the indicated vector and, at 12 h posttransfection, cells were again blocked with 2,5 mM thymidine for another 12 h to arrest all cells at the beginning of S phase. The cells were released from the thymidine block by three washes in fresh medium and allowed to progress through G 1 and into S phase by stimulation with 10% FCS. The cell cycle positioning of serum-stimulated cells was determined at different time points by propidium iodide staining and flow cytometry analysis as described above.