Cytoprotective effects of pretreating cells with WA prior to a thermal challenge.

<p>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.</p

Effect of WA on BiP, GRP94, HSP30 and HSP70 accumulation.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#s2" target="_blank">Material and methods</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#s2" target="_blank">Materials and methods</a>. 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.</p

Effect of mild heat shock on WA-induced BiP, GRP94, HSP30 and HSP70 accumulation.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#s2" target="_blank">Materials and methods</a> and in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#pone-0050547-g003" target="_blank">Figure 3</a>. 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.</p

Effect of WA on the relative levels of <i>bip</i>, <i>hsp70, hsp30</i> and <i>eIF1α</i> mRNA.

<p>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 <i>bip</i>, <i>hsp70, hsp30</i> and <i>eIF1α</i> antisense riboprobes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#s2" target="_blank">Materials and methods</a>. B) Image J software was used to perform densitometric analysis of the signal intensity for <i>bip</i> (grey), <i>hsp70</i> (black) and <i>hsp30</i> (white) mRNA levels of northern blot images as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#s2" target="_blank">Materials and methods</a>. The data are expressed as a percentage of the maximum signal (30 µM MG132 for <i>hsp70</i> and <i>hsp30</i> mRNA and 7 µM A23187 for <i>bip</i> 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.</p

Intracellular localization of BiP in response to A23187, WA and MG132.

<p>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.</p

Localization of WA-induced HSP30 accumulation.

<p>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.</p

Effect of WA on relative levels of ubiquitinated protein and proteasomal chymotrypsin-like activity.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#s2" target="_blank">Materials and methods</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#s2" target="_blank">Materials and methods</a>. 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 * (<i>p</i><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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#s2" target="_blank">Materials and methods</a>. 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 * (<i>p</i><0.05). These data are representative of three separate experiments.</p

H₂O₂ increases PDGFβ receptor phosphorylation in SH-SY5Y cells and primary neuron cultures.

<p>(A) SH-SY5Y cells were treated with vehicle (VEH) or 0.01 to 100 µM H₂O₂ for 5 min. Following drug treatments, cell lysates were evaluated by Western blot analysis as described in Materials and Methods. Data were normalized to total PDGFRβ protein expression and are expressed as the fold change (average ± S.E.M.) in phospho-1021 immunoreactivity compared to vehicle-treated cells. Representative blots for phospho-PDGFRβ 1021 (pY1021) and PDGFRβ at 180 kDa are shown. (B) Primary mouse cortical neuron cultures were treated with 0.1 µM H₂O₂ for 5 min. Lysates were evaluated for phospho-Y1021 as described in “A”. (C) SH-SY5Y cell cultures were pretreated with vehicle or 1000 µM of the ROS scavenger <i>N</i>-acetyl-l-cysteine (NAC) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min. (Data are representative of 4-6 independent experiments. * = p < 0.05 compared to vehicle-treated cells; # = p < 0.05 compared to 5-HT-treated cells, one-way ANOVA, Tukey post-test, or Student’s t-test).</p

H₂O₂ concentrations sufficient for inducing PDGFβ receptor phosphorylation do not result in cell death.

<p>SH-SY5Y cells were treated with 0, 0.1, 1, 10, 100, or 1000 µM H₂O₂ for (A) 30 min, or (B) overnight. Following treatment with MTT reagents and lysis, cell viability was measured and compared to control (VEH) values. (Data are representative of 4 independent experiments. * = p < 0.05 compared to vehicle-treated cells, one-way ANOVA, Tukey post-test).</p

5-HT can transactivate TrkB receptors via ROS.

<p>(A) SH-SY5Y cells were treated with vehicle (VEH) or 0.01 to 10 µM H₂O₂ for 5 min. Following drug treatments, cell lysates were evaluated by Western blot analysis as described in Materials and Methods. Data were normalized to total TrkB protein expression and are expressed as the fold change (average ± S.E.M.) in TrkB phospho-816 immunoreactivity compared to vehicle-treated cells. Representative blots for phospho-TrkB Y816 (pY816) and TrkB at 145 kDa are shown. (B) Cell cultures were incubated with 0.1 µM 5-HT for 0, 1, 2, 5, 10, or 15 min, and fold change in TrkB Y816 phosphorylation was measured with respect to vehicle. (C) Cultures were pretreated with vehicle or 1000 µM of the ROS scavenger <i>N</i>-acetyl-l-cysteine (NAC) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min. Normalized data was analyzed for phospho-TrkB Y816. (D) Cells were incubated overnight with 0.01 or 0.1 µg/mL pertussis toxin (Ptx) followed by 5 min treatment with 0.1 µM 5-HT. (E) Cell cultures were pretreated with vehicle or 1 or 10 µM of the PDGF receptor kinase inhibitor AG 1296 for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min. Western blots were evaluated for changes in phospho-TrkB Y816. (Data are representative of 5-6 independent experiments. * = p < 0.05 compared to vehicle-treated cells; # = p < 0.05 compared to 5-HT-treated cells, one-way ANOVA, Tukey post-test).</p