Khan S , Rammeloo AW , Heikkila JJ .

             response to heat
             protein ubiquitination
             filamentous actin
             response to endoplasmic reticulum stress

	Figure 2. Effect of WA on the relative levels of bip, hsp70, hsp30 and eIF1α mRNA.A) Cells were treated with either 5 µM WA for 16 h or 7 µM A23187, 30 µM MG132 or the appropriate volume of the DMSO vehicle (C) for 24 h at 22°C. Cells were harvested and total RNA was isolated. Total RNA (10 µg) was analyzed by northern hybridization analysis using bip, hsp70, hsp30 and eIF1α antisense riboprobes as described in Materials and methods. B) Image J software was used to perform densitometric analysis of the signal intensity for bip (grey), hsp70 (black) and hsp30 (white) mRNA levels of northern blot images as described in Materials and methods. The data are expressed as a percentage of the maximum signal (30 µM MG132 for hsp70 and hsp30 mRNA and 7 µM A23187 for bip mRNA). The standard error is represented by vertical error bars. The level of significance of the differences between samples was calculated by one-way ANOVA with a Tukey’s post-test. Significant differences between the control cells and treated cells are indicated as * (p<0.05). These data are representative of three separate experiments.
	Figure 3. Effect of WA on BiP, GRP94, HSP30 and HSP70 accumulation.A) Cells were treated with 5 µM WA for 16 h, 7 µM A23187, 30 µM MG132 or the appropriate volume of the DMSO vehicle (C) for 24 h at 22°C. Cells were harvested and total protein was isolated. Forty µg of the different protein samples were analyzed by Western blot analysis using anti-BiP, anti-GRP94, anti-HSP30, anti-HSP70, anti-AKT or anti-actin antibodies as described in Material and methods. Image J software was used to perform densitometric analysis of the signal intensity for BiP, GRP94, AKT, HSP30 and HSP70 protein bands of western blot images as described in Materials and methods. The data are expressed for each treatment as a ratio to control levels (for BiP, GRP94 and AKT accumulation) or as percentage of the maximum signal (30 µM MG132 for HSP30 and HSP70 accumulation). The standard error is represented by vertical error bars. The level of significance of the differences between samples was calculated by one-way ANOVA with a Tukey’s post-test. Significant differences between the control cells and treated cells are indicated as * (p<0.05) or Δ (p<0.10). These data are representative of three separate experiments.
	Figure 4. Temporal pattern of WA-induced BiP, GRP94, HSP30 and HSP70 accumulation.Cells were exposed to 5 µM WA (A, B) or 30 µM MG132 (C, D) for time intervals ranging from 2 to 24 h. Cells were harvested and total protein was isolated. The different protein samples were then analyzed by immunoblot analysis. Image J software was used to perform densitometric analysis of the signal intensity for BiP (white), GRP94 (black), HSP30 (white) and HSP70 (black) protein bands of western blot images as described in Materials and methods and in the legend of Figure 3. The data are expressed for each treatment as a ratio to control levels for BiP and GRP94 accumulation (panels A & C) or as a percentage of the maximum band for HSP30 and HSP70 accumulation (panels B & D). Significant differences between the control cells and WA or MG132 treated cells are indicated as * (p<0.05) or Δ (p<0.10). These data are representative of three separate experiments.
	Figure 5. Effect of mild heat shock on WA-induced BiP, GRP94, HSP30 and HSP70 accumulation.A) Cells were exposed to 2 or 5 µM WA at 22°C or 30°C for 8 h. Following treatment, cells were harvested and total protein was subjected to immunoblot analysis. B & C) Image J software was used to perform densitometric analysis of the signal intensity for BiP, GRP94, HSP30 and HSP70 protein bands of western blot images as described in Materials and methods and in the legend for Figure 3. The data are expressed for each treatment as a ratio to control levels (for BiP and GRP94 accumulation) or as percentage of the maximum signal (combined WA and mild heat shock treatment for HSP30 and HSP70 accumulation). Significant differences between the control cells and treated cells are indicated as * (p<0.05). These data are representative of three separate experiments.
	Figure 6. Intracellular localization of BiP in response to A23187, WA and MG132.Cells were grown on glass coverslips and were treated with 5 µM WA for 16 h, 7 µM A23187 or 30 µM MG132 for 24 h at 22°C. BiP was indirectly detected with an anti-BiP antibody and a secondary antibody conjugated to Alexa-488 (green). Actin and nuclei were stained directly with phalloidin conjugated to TRITC (red) and with DAPI (blue), respectively. From left to right the columns display fluorescence detection channels for actin, BiP and merger of actin, DAPI and BiP. The white arrows indicate perinuclear localization while the yellow arrows show the presence of BiP near the cell membrane. The 20-µm white scale bars are indicated at the bottom right section of each panel. These results are representative of 3 different experiments.
	Figure 7. Localization of WA-induced HSP30 accumulation.Cells were cultured on glass coverslips and incubated with 5 µM WA for 8 or 18 h or 30 µM MG132 for 24 h at 22°C. HSP30 was indirectly detected with an anti-HSP30 antibody and a secondary antibody conjugated to Alexa-488 (green). Actin and nuclei were stained directly with phalloidin conjugated to TRITC (red) and with DAPI (blue), respectively. In the 18 h set of images (bottom row), the white arrows indicate examples of structures that display co-localization of both actin and HSP30. The 5 or 20 µM white scale bars are indicated at the bottom right section of each panel. These data are representative of three separate experiments.
	Figure 8. Cytoprotective effects of pretreating cells with WA prior to a thermal challenge.Cells were maintained at 22°C in the presence of the appropriate volume of the DMSO vehicle (C; top row) or exposed to 2 µM WA for 6 h (second row) or a 37°C thermal challenge for 1 h (third row) followed by a 6 h recovery period at 22°C. Additionally, some cells were exposed to 2 µM WA for 6 h followed by a 12 h recovery at 22°C in WA-free media prior to a 37°C heat shock for 1 h with a 6 h recovery at 22°C (bottom row). A) BiP was indirectly detected with an anti-BiP antibody and a secondary antibody conjugated to Alexa-488 (green). Actin and nuclei were stained directly with phalloidin conjugated to TRITC (red) and DAPI (blue), respectively. From left to right the columns display fluorescence detection channels for actin, BiP and merger of actin, DAPI and BiP. The 20 µM white scale bars are indicated at the bottom right section of each panel. B) HSP30 was indirectly detected with an anti-HSP30 antibody and a secondary antibody conjugated to Alexa-488 (green). Actin and nuclei were stained directly with phalloidin conjugated to TRITC (red) and DAPI (blue), respectively. The 20 µM white scale bars are indicated at the bottom right section of each panel. These data are representative of 3 separate experiments.
	Figure 1. Effect of WA on relative levels of ubiquitinated protein and proteasomal chymotrypsin-like activity.A) Cells were treated with either 5 µM withaferin A (WA) for 16 h or with 7 µM A23187, 30 µM MG132 or the appropriate volume of the DMSO vehicle (C) for 24 h at 22°C. Isolated protein was subjected to immunoblot analysis employing a mouse anti-ubiquitin monoclonal antibody as described in Materials and methods. The positions of molecular mass standards in kDa are shown in the first lane (M). B) Image J software was used to perform densitometric analysis of the signal intensity for ubiquitinated protein bands of western blot images as described in Materials and methods. The data are expressed as a percentage of the lane having maximum density (MG132) while the standard error is represented by vertical error bars. The level of significance of the differences between samples was calculated by one-way ANOVA with a Tukey’s post-test. Significant differences between the control cells and A6 cells treated with 5 µM WA or 30 µM MG132 are indicated as * (p<0.05). These data are representative of at least three separate experiments. C) Effect of WA on proteasomal chymotrypsin (CT)-like activity of A6 cells. Cells were treated with WA, A23187 or MG132 as indicated above. A cell-based proteasomal CT-like assay was used to monitor the proteolytic activity as described in Materials and methods. The CT-like activity was measured and expressed as a percentage of the CT-like activity observed in control cells. The level of significance of the differences between samples was calculated by one-way ANOVA with a Tukey’s post-test. Significant differences between control cells and cells treated with WA, A23187 or MG132 are indicated as * (p<0.05). These data are representative of three separate experiments.

Abdulle, Xenopus small heat shock proteins, Hsp30C and Hsp30D, maintain heat- and chemically denatured luciferase in a folding-competent state. 2002, Pubmed, Xenbase

Abdulle, Xenopus small heat shock proteins, Hsp30C and Hsp30D, maintain heat- and chemically denatured luciferase in a folding-competent state. 2002, Pubmed , Xenbase
Arrigo, Small stress proteins: chaperones that act as regulators of intracellular redox state and programmed cell death. 1998, Pubmed
Balch, Adapting proteostasis for disease intervention. 2008, Pubmed
Bargagna-Mohan, Withaferin A targets intermediate filaments glial fibrillary acidic protein and vimentin in a model of retinal gliosis. 2010, Pubmed
Bonelli, Proteasome inhibition increases HuR level, restores heat-inducible HSP72 expression and thermotolerance in WI-38 senescent human fibroblasts. 2004, Pubmed
Borm, Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization. 2005, Pubmed
Bova, Mutation R120G in alphaB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function. 1999, Pubmed
Buchner, Purification and characterization of small heat shock proteins. 1998, Pubmed
Bush, Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance. 1997, Pubmed
Choi, Endoplasmic reticulum stress mediates withaferin A-induced apoptosis in human renal carcinoma cells. 2011, Pubmed
Devi, In vivo growth inhibitory and radiosensitizing effects of withaferin A on mouse Ehrlich ascites carcinoma. 1995, Pubmed
Doshi, The role of Hsp27 and actin in the regulation of movement in human cancer cells responding to heat shock. 2009, Pubmed
Fernando, Mutation or deletion of the C-terminal tail affects the function and structure of Xenopus laevis small heat shock protein, hsp30. 2002, Pubmed , Xenbase
Fernando, Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity. 2000, Pubmed , Xenbase
Fernando, Phosphorylation-dependent structural alterations in the small hsp30 chaperone are associated with cellular recovery. 2003, Pubmed , Xenbase
García-Mata, Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera. 1999, Pubmed
Gauley, Intracellular localization of the heat shock protein, HSP110, in Xenopus laevis A6 kidney epithelial cells. 2008, Pubmed , Xenbase
Gellalchew, Intracellular localization of Xenopus small heat shock protein, hsp30, in A6 kidney epithelial cells. 2005, Pubmed , Xenbase
Haas, Proteasome function is required to maintain muscle cellular architecture. 2007, Pubmed
Heikkila, Heat shock protein gene expression and function in amphibian model systems. 2010, Pubmed
Irobi, Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy. 2004, Pubmed
Johnston, Aggresomes: a cellular response to misfolded proteins. 1998, Pubmed
Kawazoe, Proteasome inhibition leads to the activation of all members of the heat-shock-factor family. 1998, Pubmed
Khan, Curcumin-induced inhibition of proteasomal activity, enhanced HSP accumulation and the acquisition of thermotolerance in Xenopus laevis A6 cells. 2011, Pubmed , Xenbase
Kim, Proteasome inhibitors MG132 and lactacystin hyperphosphorylate HSF1 and induce hsp70 and hsp27 expression. 1999, Pubmed
Kitiphongspattana, Proteasome inhibition alters glucose-stimulated (pro)insulin secretion and turnover in pancreatic {beta}-cells. 2005, Pubmed
Krone, Comparison of regulatory and structural regions of the Xenopus laevis small heat-shock protein-encoding gene family. 1992, Pubmed , Xenbase
Lang, Stress-induced, tissue-specific enrichment of hsp70 mRNA accumulation in Xenopus laevis embryos. 2000, Pubmed , Xenbase
Lang, Spatial pattern of constitutive and heat shock-induced expression of the small heat shock protein gene family, Hsp30, in Xenopus laevis tailbud embryos. 1999, Pubmed , Xenbase
Lee, Proteasome inhibitors cause induction of heat shock proteins and trehalose, which together confer thermotolerance in Saccharomyces cerevisiae. 1998, Pubmed
Lehman, The ubiquitin proteasome system in neuropathology. 2009, Pubmed
Liao, Proteasome inhibition induces differential heat shock protein response but not unfolded protein response in HepG2 cells. 2006, Pubmed
Lundgren, Identification and characterization of a Drosophila proteasome regulatory network. 2005, Pubmed
Lüss, A proteasome inhibitor confers cardioprotection. 2002, Pubmed
MacRae, Structure and function of small heat shock/alpha-crystallin proteins: established concepts and emerging ideas. 2000, Pubmed
Maitra, Inhibition of NFkappaB by the natural product Withaferin A in cellular models of Cystic Fibrosis inflammation. 2009, Pubmed
Malhi, Endoplasmic reticulum stress in liver disease. 2011, Pubmed
Mandal, Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade. 2008, Pubmed
Manning, AKT/PKB signaling: navigating downstream. 2007, Pubmed
Manwell, Examination of KNK437- and quercetin-mediated inhibition of heat shock-induced heat shock protein gene expression in Xenopus laevis cultured cells. 2007, Pubmed , Xenbase
Mishra, Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review. 2000, Pubmed
Miskovic, Isolation and characterization of a cDNA encoding a Xenopus immunoglobulin binding protein, BiP (grp78). 1997, Pubmed , Xenbase
Miskovic, Constitutive and stress-inducible expression of the endoplasmic reticulum heat shock protein 70 gene family member, immunoglobulin-binding protein (BiP), during Xenopus laevis early development. 1999, Pubmed , Xenbase
Mohan, Withaferin A is a potent inhibitor of angiogenesis. 2004, Pubmed
Morimoto, Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. 1998, Pubmed
Morimoto, Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging. 2008, Pubmed
Noonan, Surface expression of Hsp70B' in response to proteasome inhibition in human colon cells. 2008, Pubmed
Ohtsuka, Cytoskeletal thermotolerance in NRK cells. 1993, Pubmed
Ovsenek, DNA sequence-specific binding activity of the heat-shock transcription factor is heat-inducible before the midblastula transition of early Xenopus development. 1990, Pubmed , Xenbase
Quinlan, Fatal attraction: when chaperone turns harlot. 1999, Pubmed
Ross, The ubiquitin-proteasome pathway in Parkinson's disease and other neurodegenerative diseases. 2004, Pubmed
Santagata, Using the heat-shock response to discover anticancer compounds that target protein homeostasis. 2012, Pubmed
Sun, Localization of GRP78 to mitochondria under the unfolded protein response. 2006, Pubmed
Taylor, Toxic proteins in neurodegenerative disease. 2002, Pubmed
Tonkiss, Regulation of heat shock gene transcription in neuronal cells. 2005, Pubmed
Van Montfort, Structure and function of the small heat shock protein/alpha-crystallin family of molecular chaperones. 2001, Pubmed
Voellmy, On mechanisms that control heat shock transcription factor activity in metazoan cells. 2004, Pubmed
Voyer, Comparison of the effect of heat shock factor inhibitor, KNK437, on heat shock- and chemical stress-induced hsp30 gene expression in Xenopus laevis A6 cells. 2008, Pubmed , Xenbase
Walcott, Celastrol can inhibit proteasome activity and upregulate the expression of heat shock protein genes, hsp30 and hsp70, in Xenopus laevis A6 cells. 2010, Pubmed , Xenbase
Westerheide, Heat shock response modulators as therapeutic tools for diseases of protein conformation. 2005, Pubmed
Wiegant, Stress-induced thermotolerance of the cytoskeleton of mouse neuroblastoma N2A cells and rat Reuber H35 hepatoma cells. 1987, Pubmed
Woolfson, Examination of cadmium-induced expression of the small heat shock protein gene, hsp30, in Xenopus laevis A6 kidney epithelial cells. 2009, Pubmed , Xenbase
Wu, mTOR complex 2 targets Akt for proteasomal degradation via phosphorylation at the hydrophobic motif. 2011, Pubmed
Xu, 2,3-Dihydrowithaferin A-3beta-O-sulfate, a new potential prodrug of withaferin A from aeroponically grown Withania somnifera. 2009, Pubmed
Yang, The tumor proteasome is a primary target for the natural anticancer compound Withaferin A isolated from "Indian winter cherry". 2007, Pubmed
Yang, Natural compounds with proteasome inhibitory activity for cancer prevention and treatment. 2008, Pubmed
Young, Simultaneous exposure of Xenopus A6 kidney epithelial cells to concurrent mild sodium arsenite and heat stress results in enhanced hsp30 and hsp70 gene expression and the acquisition of thermotolerance. 2009, Pubmed , Xenbase
Young, Proteasome inhibition induces hsp30 and hsp70 gene expression as well as the acquisition of thermotolerance in Xenopus laevis A6 cells. 2010, Pubmed , Xenbase
Yu, Withaferin A targets heat shock protein 90 in pancreatic cancer cells. 2010, Pubmed