EDTA, 200 mM NaCl, 0.2% SDS, and 100 mg of proteinase K at 55uC. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with isopropanol. Genomic PCR was performed by using two primer sets (set 1: forward primer, 59- ATGGCTTCTAGCTATCCTTATG-39, and reverse primer, 59- GTAGCAACCTGTGCAATTTCTTC-39; set 2: forward primer, 59-CTGCTAACCATGTTCATGCC-39 and reverse rimer, 59- ACAAGTTTAGCTCCAATGTTTTTGTA-39). All the experi- ments were performed with 4-week-old males. Analysis of apoptotic cells in DEN-induced liver damage The four-week-old male mice were injected intravenously with phosphate-buffered saline (PBS, pH 7.4) or TNF-a (6 mg/kg) via the lateral tail vein 6 hrs before intraperitoneal administration of DEN (10 mg/kg). After 48 hrs of DEN treatment, animals were sacrificed and rapidly perfused with PBS followed by 4% paraformaldehyde. The livers were removed and frozen in OCT embedding medium, and then a series of tissue sections (10 mm in thickness) was obtained in cryostat (Leica). The sections were incubated in 50 ml of terminal deoxynucleotidyl transferase-mediate uridine 59-triphosphate-biotin nick-end labeling (TUNEL) fluorescent reaction mixture (In situ Cell Death Detection Kit, Roche Diagnostics) for 60 mins at 37uC in a dark chamber, washed and subsequently counterstained with 49,69-diamidino-2-phenylindole (DAPI, 1 mg/ml, Sigma) for 30 mins. The sections were mounted using the Vectashield mounting medium and examined using a LSM510 confocal laser- scanning microscope (Carl Zeiss, Germany). TUNEL-positive cells were counted and averaged from three tissue sections per mouse. All animal experiments were performed in compliance with the institutional guidelines (Ewha Womans University, Korea) for the care and use of laboratory animals. In vitro osteoclastogenesis The non-adherent bone marrow-derived monocytes/macrophag- es (BMM) lineage cells derived from C57BL/6 mice were seeded and cultured in a-MEM (Invitrogen) containing 10% FBS and M-CSF (10 ng/ml, R&D systems). After 2 days, the nonadherent cells including lymphocytes were removed and the adherent cells were used as BMMs. The differentiation of BMM to osteoclast cells was induced by treating them with either soluble RANKL (50 ng/ml, Peprotech) or TNF-a (20 ng/ml) in the presence of M-CSF. After 5 days of induction, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). The cells were observed using a Zeiss Axiovert 200 microscope (Carl Zeiss) equipped with a plan-Neofluor objective lens. The images were analyzed using AxioVision 3.1 software (Carl Zeiss). The TRAP-positive multinucleated (.3 nuclei) cells were counted as osteoclast-like cells. Statistics Data were analyzed using Student’s t-test on SigmaPlot 8.0 software. The P values were derived to assess statistical significance and indicated on figure panels. Supporting Information Figure S1 Action of Hsp60-specific antisense oligodeoxynucleo- tide (AS-ODN). A. Schematic representation of two different Hsp60 AS-ODNs. B. Hsp60 expression in mock- or ODN- transfected HeLa cells. C. TNF-a-induced MAP kinase activation in HeLa cells transfected with either mock or ODNs. The activation was analyzed by using phospho-specific antibodies. The phosphoblots were re-probed with whole protein antibodies for equal loading. D. Activation of various transcription factors in mock- or ODN-transfected HeLa cells. AP-1 and NF-AT Hsp60 for IKK Regulation transcriptional activation were induced by epidermal growth factor (EGF, 100 ng/ml). CRE transcriptional activation was induced by forskolin (1 mM). E. TNF-a-induced NF-kB transcrip- tional activation in 293T and A549 cells transfected with either mock or ODNs as indicated. In D and E, the relative luciferase activity was measured using an enhanced luciferase assay kit (Promega) and normalized to the b-galactosidase activity. Data are means 6 S.D. of four independent experiments (In E, *P,0.001 and **P,0.05 versus stimulated S-ODN-transfected cells). Found at: doi:10.1371/journal.pone.0009422.s001 (2.28 MB EPS) Figure S2 Selective role of cytosolic Hsp60 (Hsp60c) in IKK/NF- kB signaling. A. TNF-a-induced JNK activation in HeLa cells expressing Hsp60c (HA tag). B - D. Activation of various transcription factors in HeLa cells transfected with either control vector or Hsp60c (HA tag). AP-1 (B) and NF-AT (C) transcriptional activation were induced by epidermal growth factor (EGF, 100 ng/ml). CRE transcriptional activation (D) was induced by forskolin (1 mM). The relative luciferase activity was measured using an enhanced luciferase assay kit (Promega) and normalized to the b-galactosidase activity. Data are means 6 S.D. of three independent experiments. Found at: doi:10.1371/journal.pone.0009422.s002 (1.35 MB EPS) Figure S3 DEN induces hepatic cell apoptosis. The four-week- old C57BL/6j male mice were intraperitoneally injected with DEN (10 mg/kg). After the indicated time periods of DEN treatment, animals were sacrificed and processed to prepare tissue sections and images as described in Experimental Procedures. TUNEL positive cells were counted in three tissue sections per mouse. Representative images (A) are shown. Data in the quantitative graph (B) are mean 6 S.D. of TUNEL positive cells per unit area. Found at: doi:10.1371/journal.pone.0009422.s003 (4.30 MB EPS) Figure S4 Mitochondrial release of Hsp60 in staurosporine- treated HeLa cells. HeLa cells were treated with 1 mM staurosporine and then subjected to subcellular fractionation using the ProteoExtract subcellular proteome extraction kit (Roche). Peroxiredoxin-III (Prx III) and a-tubulin were used as mitochon- drial and cytosolic markers, respectively. In particular, an antioxidant enzyme called Prx III (25-kDa in molecular size), which is present in the mitochondrial matrix, was useful for monitoring mitochondrial rupture. In western blots, the mito- chondrial fractions were loaded at the volume of one-fifth of cytosolic fraction for appropriate comparison. Found at: doi:10.1371/journal.pone.0009422.s004 (1.31 MB EPS) Figure S5 Cytosolic Hsp60 plays a significant survival role in RANK-mediated osteoclastogenesis. The ODN-pretreated BMM cells were treated with either RANKL (A) or TNF-a (B) for 5 days in the presence of M-CSF. The TRAP-positive multinucleated osteoclast cells were counted as described in the Experimental Procedures. Data represent the means6SD of triplicate from one of two independent sets of experiments, all of which showed similar results (* P,0.02 versus the stimulated sense-ODN). Representative pictures are shown. Found at: doi:10.1371/journal.pone.0009422.s005 (7.72 MB EPS) Acknowledgments We are grateful to M. Karin for providing IKKa/b2/2 3T3 cells, F. Mercurio for sharing Flag-IKKa/b-encoding plasmids, and W. Herr for sharing pCGN plasmid vector. We are also grateful to M. S. Lee, T. S. Chang, and G. T. Oh for helping us with IKK 3T3 cell lines, ASK1 assay, and transgenic mice generation, respectively. Finally, we extend our gratitude to members in core research facilities for laboratory animals and mass spectrometry for technical assistance. PLoS ONE | www.plosone.org 14 March 2010 | Volume 5 | Issue 3 | e9422