STAT3 and eIF4E [7,21,22] in cells devoid of Hsp27. Hence, despite Hsp27 has no ATPase box and no chemical inhibitors are yet available, we tested whether this chaperone could also regulate a set of client proteins. Using shRNA-mediated depletion, co-immunoprecipitation and protein activity assays, we show here that, in unstressed HeLa cells, Hsp27 is associated with three putative client proteins: histone deacetylase 6 (HDAC6), signal transducer and activator of transcription 2 (STAT2) and procaspase-3; three polypeptides that play major roles in cytoskeleton deacetylation, signal transduction and apoptosis. Experimental Procedures Cell culture and transfections All cells were purchased from the ATCC Cell Biology Collection and were grown at 37uC in a humidified atmosphere containing 5% CO2. HeLa or MCF-7 cells were grown in Dubelcco’s modified Eagle’s medium (DMEM) supplemented with 10% heat inactivated fetal calf serum. For transient expression, one day before transfection with the appropriate DNA vector, exponentially growing cells were seeded at a density of 1.56106 cells/78 cm 2 . According to the LipofectamineTM reagent proce- dure (Invitrogen, Cergy Pontoise, France) DNA vector was left on cells for 3 h. Thereafter, cells were washed once with PBS before being further incubated in fresh culture medium. Forty-eight hours after transfection, cells were submitted to the different treatments. Gel electrophoresis and immunoblotting After treatment, cells were immediately rinsed twice in ice-cold PBS and scraped off the dish. At this point, aliquots were withdrawn for determination of protein concentration. Thereafter, cells were lysed in boiling SDS buffer (62.5 mM Tris-HCl, pH 6.8; 1% SDS; 0.1 M dithioerythritol; 0.001% bromophenol blue and 10% glycerol). Cell lysates were subjected to SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) performed as previously de- scribed [23]. The detection of immunoblots was performed with the ECLTM system (Amersham Life Science, Pantin, France). Autoradiographs were recorded on X-Omat LS films (Eastman Kodak Co, Rochester, NY). Sizing Chromatography HeLa or MCF-7 cells used to prepare cytosolic supernatants for sizing chromatography experiments were grown as detailed above. Cells from five 100-mm culture plates were harvested on ice by scraping and spun (10006g, 5 min, 4uC). They were then washed and lysed in the column equilibration buffer (20 mM Tris, pH 7.4; 5 mM MgCl2; 20 mM NaCl; 0.1 mM EDTA) supplemented with 0.1% Triton X-100. Cell lysates were spun (10,0006g, 10 min) and supernatants were loaded on a sepharose CL-6B column (Sigma, St Louis, MO). Columns fractions were analyzed by immunoblotting as previously described [6]. Molecular-mass markers used to calibrate the gel-filtration column included carbonic anhydrase (29 kDa), albumin (66 kDa), alcohol dehy- drogenase (150 kDa), b-amylase (200 kDa), apoferritin (440 kDa), thyroglobulin (669 kDa) and Dextran Blue (.2000 kDa) (Sigma, St Louis, MO). Co-Immunoprecipitation experiments (Co-IP) Co-IP experiments were performed with samples from the sizing columns (see above) known to contain the targeted polypeptides. 2 ml samples of the pooled column fractions were incubated (1 h, 4uC) with Hsp27 antibody (goat polyclonal anti- Hsp27 antibody, Santa Cruz Biotechnologies-Clinisciences, Mon- STAT2, Caspase3 and HDAC6 as Hsp27 Client Proteins trouge, France) followed by incubation (1 h, 4uC) with protein G sepharose (50 ml of a 50% bead slurry per sample, GE Healthcare, Ve´lizy, France). Samples were briefly centrifuged (5,0006g, 30 s), supernatants were discarded and bound proteins were eluted from beads. Detection of co-immunoprecipitated proteins was per- formed in immunoblots probed with the corresponding antibodies. ShRNA construction The pSuperNeo plasmid (Oligoengine, Madison, WI) was used for DNA vector-based shRNA construction. Based on the cDNA of Hsp27 (HUGO gene nomenclature committee accession No. HGNC:5246) and the shRNA designing tool provided freely by Ambion at its website (http://www.ambion.com), we synthe- sized DNA templates encoding Hsp27-specific and control shRNAs. The targeted oligonucleotide sequence was: 59- GCTGCAAAATCCGATGAG-39. After annealing, ligation and transformation into competent DH5a bacteria (Invitrogen, Ill- Kirch, France), positive colonies were selected through their antibiotic resistance. The correct sequences of the final DNA preparations were confirmed by sequencing (GenomExpress, Meylan, France). pSuperNeo-MsRNA27 and pSuperNeo- ScRNA27 (scramble, Sc27) were designed respectively as degen- erated (Ms) and scramble (Sc) controls from the above mentioned sequence of Sh27. pCI-Neo vector was used as a positive control of cell transfection. ShRNA-HDAC6 and its random controls will be described elsewhere. Generation of stable HeLa and MCF-7 cells depleted in Hsp27 Five cell culture dishes were seeded at 16105 cells/78 cm2. Transfection of HeLa and MCF-7 cells was performed as described above. One day later, positively transfected cells were selected by neomycin at a concentration of 0.5 (HeLa) or 1 (MCF- 7) mg/ml. The level of Hsp27 in neomycin resistant clones was then tested by immunobloting. Five independent clones of each cell line expressing reduced levels of Hsp27, named: HSh27 (HeLa) and MSh27 (MCF-7) and four independent mismatch control clones expressing normal levels of Hsp27 (named: HMs27 and MMs27) were selected, propagated and analyzed. Determination of STAT2 transcriptional activity The different cell lines were transiently transfected with a STAT1/STAT2-responsive luciferase construct encoding the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the interferon stimulated response element (ISRE) (SABiosciences, Frederick, MD, USA). Cells were treated 6 h with 50 U/mL interferon-a to activate the STAT2 interferon response. In this experiment, Hsp27 phosphorylation was inhibited by SB203580 and SB202190 (p38MAPkinase and MK2 inhibitors, respectively) (Sigma, St Louis, MO). Quantitative PCR To assay Caspase3, HDAC6, STAT2 and 3 expression, 1 mg of TRIzol extracted total RNA was reverse-transcribed using the SuperScriptIII kit (Invitrogen, Cergy Pontoise, France). Real-time quantitative PCR was performed on the MX-3000P cycler (Agilent, Massy, France) using QuantiTect SYBR Green kit and QuantiTect Primer Assays (Qiagen, Courtaboeuf, France). All reactions were run in triplicate and gene expression levels were normalized to the ribosomal gene RPS17 using the DDCt method. The sequences of the primers are available upon request. PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e29719