Plasmids and DNA Constructs
The enhanced yellow fluorescent protein (YFP) or cyan-fluorescent protein (CFP) were used to construct fluorescent-tagged versions of PML, RNF4, or SUMO-2 (See Table S1 in Supplemental Material). Human PMLIII (accession number AAB19601
) or rat RNF4 (accession number NM_019182
) were amplified by PCR from pcDNA3.1 to generate SalI and BamHI sites flanking the sequence. PCR products were then digested with the appropriate enzymes and inserted in place of H2B into either pBOS-H2B-YFP or pBOS-H2B-CFP vectors (Leung et al., 2004
). These vectors contain the mammalian EF1α promoter to drive expression of the fluorescent-tagged protein and harbor the blasticidin resistance gene as a selection marker. Fluorescent-tagged RNF4 mutants (RNF4-CS1 and RNF4 mtSIM1, 2, 3, 4) were obtained according to the strategy described above. Human SUMO-2 full-length (accession number CAG46970
) was amplified by PCR to generate EcoRI and BamHI sites flanking the sequence. PCR product was inserted in the C-terminal region of EGFP into the pEGFP-C1 plasmid (Clontech, Palo Alto, CA).
Antigen affinity purified sheep SUMO-1, sheep SUMO-2, chicken RNF4, and chicken PML antibodies were prepared in house. Mouse anti–β-actin (Sigma, St. Louis, MO) and mouse anti-GFP (clones 7.1 and 13.1, Roche, Indianapolis, IN) were obtained from commercial sources. Rabbit anti-RNF4, mouse anti-PML 5E10, rabbit anti-PML, mouse anti-p53 (DO-1), and mouse anti-nucleolin antibodies were a kind gifts from Jorma Palvimo (University of Kuopio, Finland), Roel van Driel (University of Amsterdam, The Netherlands), Anne Dejean (Institute Pasteur, Paris, France), David Lane (University of Dundee, Scotland), and Ara Hovanessian (University of Paris, France), respectively.
Cell Culture and Immunofluorescence Analysis
HeLa cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (GIBCO BRL, Grand Island, NY) in a 10% CO2 atmosphere at 37°C. To establish PML-YFP stable cell line, HeLa cells were grown in 10-cm Petri dishes to 50% of confluence and were transfected the next day with the fluorescent-tagged PML-YFP plasmid using Fugene 6 (Roche) according to the manufacturer's directions. Two days posttransfection, 1 μg/ml blasticidin was added to select cells stably expressing the fusion protein. Fluorescent cells were then sorted using a FACS Vantage SE cell sorter with DIVA option (BD Biosciences, San Jose, CA) with a helium-neon laser at 488 nm. The same protocol of transfection was used to establish the HeLa YFP-SUMO-2 cell line, except that 100 μg/ml neomycin was used to select resistant clones.
For immunofluorescence experiments, cells were seeded onto coverslips and were fixed with 4% paraformaldehyde for 10 min, at room temperature. Cells were then permeabilized with 0.2% Triton for 10 min and were blocked in PBS containing 5% BSA and 0.1% Tween for 30 min. Primary and secondary antibodies diluted in PBS with 1% BSA and 0.1% Tween were successively added on the cells for 1h. Cells were then stained 0.1 μg/ml DAPI and were mounted in Vectashield mounting medium.
Cell Cycle Analysis
Hela cells or HeLa PML-YFP cell line were trypsinized and washed with PBS before fixation with cold 70% ethanol for 2 h at 4°C. Cells were centrifuged at 1000 rpm for 5 min and washed twice in PBS. Cells were incubated in PBS containing 50 mg/ml propidium iodide (PI), 50 μg/ml RNAseA and 0.1% (vol/vol) Triton X-100 for 30 min. The intensity of DNA staining (FLH-2) was determined by flow cytometry on a FACS Calibur (Becton Dickson, San Diego, CA) to determine the percentage of cells in G1, S, and G2/M phases.
Protein Extraction and Western Blot Analysis
Cells were lysed in 2× Laemmli lysis buffer (150 mM Tris-HCL (pH 6.8), 5% (wt/vol) SDS, 25% (vol/vol) glycerol, and 0.01% (wt/vol) bromophenol blue) supplemented with 10 mM iodoacetamide. Lysates were clarified by sonication for 10 s on low power (Branson digital sonifier 450) and protein concentration was determined by using the BCA assay kit (Thermo Scientific). β-mercaptoethanol was added to a final concentration of 0.75 M into each sample. Total cellular protein was subjected to SDS-polyacrylamide gels electrophoresis (SDS-PAGE) and transferred onto Hybond membrane for Western blot analysis.
For immunoprecipitation of PML-YFP protein, nuclear extracts were prepared from HeLa cells stably expressing PML-YFP grown in 10-cm Petri dishes. After scraping into the medium, cells were washed twice in PBS containing 100 mM iodoacetamide and were collected by centrifugation at 1500 rpm for 5 min. Cells were resuspended in ice cold buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2,10 mM KCl, complete protease inhibitor cocktail tablets (Roche), 200 mM iodoacetamide) and were disrupted by using a syringe to pass them through a narrow (26 gauge) needle. Nuclei were separated from the cytoplasmic fraction by centrifugation at 3200 rpm for 5 min at 4°C, washed twice with buffer A, and resuspended in RIPA buffer (50 mM Tris pH 6.8, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 10 mM iodoacetamide). Nuclear extract was sonicated and clarified by centrifugation at 13,000 rpm for 1 min. The nuclear supernatant was precleared with Sepharose beads before incubation with GFP-trap beads (Chromotek) for 2 h at 4°C. Beads were collected by centrifugation at 3200 rpm and washed twice with RIPA buffer. Proteins were eluted by adding 2× SDS lysis buffer and analyzed by Western blotting.
Treatment of Cells with Cycloheximide
To inhibit protein synthesis, HeLa cells were grown in six-well plates for 24 h before addition of fresh medium containing 50 μg/ml cycloheximide. Cells were harvested at different times after cycloheximide treatment and subjected to SDS-PAGE and Western blot analysis to detect RNF4, p53 and actin proteins. To carry out real-time imaging, 50 μg/ml cycloheximide and 1 μM arsenic in Leibovitz's L-15 medium (Invitrogen) supplemented with 10% fetal bovine serum was added to PML-YFP cells seeded in Labtek chamber slides.
Quantitative Reverse Transcription-PCR
To quantify endogenous Rnf4 transcripts during arsenic treatment, total RNA was extracted from HeLa cells stably expressing PML-YFP using an SV total RNA isolation kit (Promega). Total cDNAs were amplified with a SuperScript II reverse transcriptase kit (Invitrogen), and real-time quantitative PCR was performed using a TaqMan system for 40 cycles of 15 s at 95°C and 1 min at 60°C with an ABI 7700 instrument (Applied Biosystems). TaqMan probes used for detection of human RNF4 and the internal control actin were purchased from Applied Biosystems (Hs00231302_m1 RNF4, Hs99999903_ml ACTB).
Live-Cell Imaging in Real Time and Measurement of Fluorescence Intensities
For live-cell imaging experiments, HeLa cells were seeded onto Lab-Tek II four-chamber slides (Nalgen Nunc) and transfected the next day with the appropriate fluorescent-tagged proteins using Fugene 6 (Roche). HeLa cells stably expressing fluorescently tagged fusion proteins were seeded in the same way. When required, double-stranded small interfering RNA (siRNA) for RNF4 or control scrambled sequence (Dharmacon, Lafayette, CO) were introduced at 5 nM final concentration using Lipofectamine RNAiMAX (Invitrogen). Two days later, cells were changed into Leibovitz's L-15 medium (Invitrogen) supplemented with 10% fetal bovine serum. Images were collected using the DeltaVision microscope system (Applied Precision) with an Olympus IX70 microscope and a cooled CCD camera (Coolsnap HQ, Photometrics). Temperature was maintained at 37°C using an environmental chamber (Solent Scientific, Segensworth, UK). Stacks of 20 sections with z-step set to 400 nm were collected in different channels with 35–50 ms exposure with a Plan Apo 60 × 1.40 numerical aperture objective lens (Olympus). A single z-section was captured by differential interference contrast microscopy (DIC) to visualize the position of PML NB within the cells during arsenic treatment. Collected images were deconvolved with SoftWoRx (Applied Precision), and z-stack projections were made before merging with the optical section. Because PML structures were mobile, a region of interest (ROI) containing one PML body has been defined at each time point to measure fluorescence intensities using OMERO Beta-4.3 software. The intensities in a random region outside the nucleoplasm divided by the region of interest was taken as the value to which the intensities within the PML nuclear body were compared. Data were collected from around 20 cells.
Fluorescence Recovery after Photobleaching
Fluorescence recovery after photobleaching (FRAP) experiments were carried out on the same slides and microscope described above. Entire PML NBs were bleached with a 488-nm argon laser at 50% laser intensity to reduce the YFP signal by 90%. A nucleoplasmic region outside PML NB was also bleached as a control. Images were taken before the bleach pulse and image acquisition after bleaching was adjusted according to the recovery of the fluorescent proteins by using 0.05% laser transmission intensity to minimize scan bleaching. As PML recovers slowly after photobleaching, 300 images were collected at 1-min intervals over a 10 min period whereas 32 images were captured for RNF4 within 11 s. Projection of z-sections were made only for PML. The fluorescence intensity was measured in bleached PML NBs and in control areas outside the nuclear dots and quantified with OMERO Beta-4.3 software. The fluorescence intensity in the bleached PML was normalized to the nonbleached signal after substraction of the background signal. Data were collected from ten PML NBs.
Fluorescence Resonance Energy Transfer Acceptor Photobleaching
Fluorescence resonance energy transfer (FRET) was measured by acceptor photobleaching method as previously described (Ellis et al., 2008
). The FRET experiment was conducted on a DeltaVision microscope system (Applied Precision) fitted with a quantifiable laser module, including a 20-mW 532-nm CW laser, suitable for photobleaching YFP without cobleaching CFP. Fluorescence of the donor (CFP) was observed by using argon laser 436/10 nm excitation and 480/40 band-pass emission filters, whereas fluorescence of acceptor (YFP) was observed using argon laser 532 nm excitation and 580/70 band-pass emission filters. Images were collected with a Plan-Apochromat 100 × 1.35 numerical aperture lens (Olympus) and a cooled CCD camera (CoolSnap HQ; Photometrics).
Photobleaching of YFP fluorescence was achieved by irradiation of the region of interest containing one PML NB with the 532 nm excitation filter at 50% laser intensity. A nucleoplasmic region of the same area was bleached as a negative control, resulting in minimal CFP bleaching (0–2%) which has not been taken into account for the calculation of FRET efficiency. CFP fluorescence was measured before (CFPbefore) and after (CFPafter) the YFP bleaching by collecting a total of 23 images. Pixel intensities of the different regions were analyzed with OMERO software and apparent FRET efficiency was expressed as the percent increase of prebleach CFP fluorescence in the region of interest compared with that observed after YFP photobleaching, according to the equation: FRET efficiency [%] = (CFPafter
) × 100/CFPafter
, previously described by Stanek et al.
(Stanek and Neugebauer, 2004
). P value was calculated with the two-tailed homoscedastic t
test comparing the FRET efficiencies with and without As2