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.

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