Hsp60 for IKK Regulation Figure 8. Loss of cytosolic Hsp60 induces cell death in response to TNF-a by inducing ROS and ASK-1 activation. A. RNase protection assay for induction of anti-apoptotic genes in ODN-transfected cells. The autoradiogram shown is a representative of three independent experiments. B and C. QPCR for induction of endogenous NF-kB target genes in ODN-transfected cells (B) and antibody-transfected cells (C) (n = 3, *P,0.01, **P,0.001). D. TNF-a-mediated production of cellular ROS in ODN-transfected cells. The representative images are shown. Data are means 6 S.D. of fold increase versus untreated mock cells of the relative DCF fluorescence (n = 4, *P,0.05, **P,0.001). E. JNK and p38 MAPK activation in ODN-transfected cells. Representative blots are shown. Data in the graphs are means 6 S.D. of the intensities of phospho-JNK (p46) or phospho-p38 bands that had been normalized by the corresponding non-phospho-bands (n = 3, *P,0.05, **P,0.001). F. ASK-1 activation in ODN-transfected cells. The kinase activity (KA) was averaged with the values from two independent experiments, and presented as a fold increase of the activity versus the unstimulated and mock- transfected cells (lane 1). G. TNF-a-induced cell death in mock or ODN-transfected HeLa. Data are means 6 S.D. (n = 3, *P,0.01). H. TNF-a-induced cell death of colon carcinoma cells transfected with ODNs. The level of Hsp60 in subcellular fractions is shown (Upper). Data in the graph are means 6 S.D. (n = 3, *P,0.05, **P,0.01). Cell death was analyzed by FACS after staining with annexin V-fluorescein isothiocynate and propidium iodide. doi:10.1371/journal.pone.0009422.g008 function of Hsp60 proteins in the extra-mitochondrial compart- ments [25]. Our study demonstrates for the first time that, by directly interacting and regulating IKK activation, the cytosolic Hsp60 serves as survival guidance controlling mitochondrial- derived ROS through NF-kB target gene expression. Therefore, either antagonizing the interaction of Hsp60 with IKK or reducing the level of the cytosolic Hsp60 could be an additional therapeutic tool in anti-inflammatory strategy. Materials and Methods Reagents Antibodies to IKKa (B-8), IKKc (FL-419), Hsp90 (H-114), Hsp60 (K-19 and N-20), IkBa (C-21), JNK1 (C-17), ASK-1 (H- 300 and F-9), glutathione S-transferase (B14), and goat IgG were purchased from Santa Cruz Biotechnology (Santa Crus, US); Anti- Flag antibody (M2) was purchased from Sigma; Anti-hexahistidine Figure 9. Cytosolic Hsp60 protects hepatic cells in vivo from stress-induced apoptosis via IKK activation. A. Schematic representation of transgenic vector containing HA-tagged human Hsp60c. B. Identification of two transgenic mouse lines (T4 and T11). Genomic PCR was performed using two different sets of PCR primers, both of which were specific to Hsp60 transgene. The transgenic vector (V) and C57BL/6j (B6) mouse genomic DNA were used as positive and negative control, respectively. C. Expression of Hsp60c protein in various tissues of transgenic mice. The tissue homogenates were immunoblotted using anti-HA antibody. D. IKK activation in the liver of control B6 mice or HA-Hsp60c-expressing transgenic mice (T4 and T11) intravenously injected with TNF-a. E and F. DEN-induced cell death in the liver of control and transgenic mice primed with or without TNF-a, as measured by TUNEL assay. The representative images (E) are shown. The quantified data in the graph (F) are means 6 S.E.M. of the number of TUNEL-positive cells per unit area (n = 3, * P,0.01 versus paired stimulated one). doi:10.1371/journal.pone.0009422.g009 PLoS ONE | www.plosone.org 11 March 2010 | Volume 5 | Issue 3 | e9422