Changes in protein expression and activation of heat shock proteins associated with HSP90 inhibition.

<p>(<b>A</b>) Quantification of HSP90α and HSP70 expression levels among the <i>Pkd1</i>^–/– groups. <i>n</i> = 6–8 mice. * indicates comparisons to <i>Pkd1</i>^–/–, vehicle-treated mice: **, <i>P</i>≤0.01; ***, <i>P</i>≤0.001. (<b>B–E</b>) Negative correlations between HSP70, HSP90α expression in the drug treatment groups and (<b>B, D</b>) cyst volume/region of interest (ROI) (<i>P</i> = 0.0046 for HSP70 and <i>P</i> = 0.0136 for HSP90) and (<b>C, E</b>) liver weight/body weight (lw/bw) ratios (<i>P</i> = 0.0141 for HSP70 <i>P</i> = 0.0138 for HSP90). Dots represent individual mice; lines represent linear regression functions. <i>n</i> = 6–8 mice.</p

HSP90α is upregulated in epithelial cells lining liver cysts.

<p>(<b>A, B</b>) Representative hematoxylin stained liver sections with immunohistochemical detection of HSP90α (brown) from three (<b>A</b>) wild type (wt) and (<b>B</b>) <i>Pkd1</i>^–/– mice. bd  =  bile duct, pv  =  portal vein, c  =  cyst. Scale bars  = 100 µm; magnification, 20x.</p

Changes in protein expression and activation of signaling proteins associated with HSP90 inhibition.

<p>(<b>A</b>) Differences in expression of phosphorylated Y¹⁰⁶⁸ and Y¹¹⁷³ EGFR and total EGFR normalized to actin, and Y¹⁰⁶⁸ and Y¹¹⁷³ EGFR normalized to total EGFR, following treatment with the indicated doses of STA-2842. *, P<0.05; **, P<0.01. (<b>B</b>) Insignificant differences in expression of phosphorylated T²⁰²/Y²⁰⁴ ERK1/2 or total ERK1/2, normalized to tubulin, and phosphorylated T²⁰²/Y²⁰⁴ ERK1/2 normalized to total ERK1/2, following treatment with the indicated doses of STA-2842; differences are not significant (<b>C</b>) Positive correlations between the ratio of phosphorylated T²⁰²/Y²⁰⁴ ERK1/2 normalized to total ERK1/2 expression in the drug treatment groups and (left) cyst volume/region of interest (ROI) (<i>P</i> = 0.174) and (right) liver weight/body weight (lw/bw) ratio (<i>P</i> = 0.040). Dots represent individual mice; lines represent linear regression functions. <i>n</i> = 6–8 mice. All data graphed as mean ± standard error of the mean (SEM). (<b>D</b>) Expression of phosphorylated T²⁰²/Y²⁰⁴ ERK1/2 in cystic epithelia (left) or total liver (right) quantified from immunohistochemical staining of liver sections from <i>Pkd1</i>^–/– mice treated with vehicle or 100 mg/kg STA-2842 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114403#pone.0114403.s004" target="_blank">Fig S4</a>). **, P<0.01. (<b>E</b>) Differences in expression of phosphorylated phS²³⁵/S²³⁶ S6 or total S6, normalized to tubulin, and phosphorylated phS²³⁵/S²³⁶ S6 normalized to total S6, following treatment with the indicated doses of STA-2842; *, P<0.05. <i>P-</i>values for comparisons between <i>Pkd1</i>^–/– vehicle- versus 100 mg/kg-treated groups bordering on significant (<i>P</i> = 0.060). (<b>F</b>) Differences in expression of phosphorylated phS⁴⁷³ AKT or total AKT, normalized to tubulin, and phosphorylated phS⁴⁷³ AKT normalized to total AKT, following treatment with the indicated doses of STA-2842; *, P<0.05.</p

Histopathological development of cysts.

<p>(<b>A</b>) Representative livers collected from <i>Pkd1</i>^–/– or <i>wt</i> mice treated with vehicle or STA-2842 at the indicated doses, at 6.5 months of age. (<b>B</b>) Liver weights (lw) to body weight (bw) ratio of in <i>wt</i> (+) or <i>Pkd1</i>^–/– (–) mice after 10 weeks of treatment with STA-2842 (50, 100) or vehicle (–). <i>n</i> = 5–8 mice. * indicates comparisons to <i>Pkd1</i>^–/–, vehicle-treated mice; # indicates comparisons to <i>wt</i> vehicle-treated mice: *, <i>P</i><0.05; ^##, <i>P</i><0.01; ^###, <i>P</i><0.001; ^####, <i>P</i><0.0001. (<b>C</b>) Representative H&E stained liver sections after 10 weeks of treatment with vehicle or drug in <i>wt</i> (+) or <i>Pkd1</i>^–/– (–) mice, indicating extent of cystogenesis. Scale bars  = 100 µm; magnification  = 10x. (<b>D</b>) Cystic indices quantified as a percentage of grid intersections that cross cysts on H&E slides. <i>n</i> = 6–8 mice. * indicates comparisons to <i>Pkd1^–/–</i>, vehicle-treated mice: *, <i>P</i><0.05. All data graphed as mean ± standard error of the mean (SEM). (<b>E</b>) Relative change in cyst burden by drug dose (6 month value divided by 4 month value), with simple linear fits of the relationship shown. None of the slopes were statistically significant (p>0.11 in all three cases). Since the range of values differed among the three doses, the X-axis is drawn on the log scale to better depict the relationships. <b>F</b>. Relative expression of cleaved PARP, normalized to total PARP, following treatment of <i>Pkd1</i>^–/– (–) mice with the indicated doses of STA-2842. **, <i>P</i><0.01 to vehicle-treated mice; ^##, P<0.01 relative to mice treated with 50 mg/kg STA-2842. <b>G</b>. Relative expression of cleaved caspase 8, normalized to GAPDH, following treatment of <i>Pkd1</i>^–/– (–) mice with the indicated doses of STA-2842. *, <i>P</i><0.05, ***, P<0.001 in reference to vehicle-treated mice.</p

HSP90 inhibition reduces late stages of liver cyst formation in conditional <i>Pkd1</i>^–/– mice.

<p>(<b>A</b>) Schematic outlining the timetable of the study. (<b>B</b>) Representative MRI images of mice, imaged at 0, 5, and 10 weeks of treatment with vehicle or drug (STA-2842) in <i>wt</i> (+) or <i>Pkd1</i>^–/– (–) mice. Scale bars  = 0.5 cm. (<b>C</b>) Cyst volume estimated as a percentage of hepatic tissue at 0, 5, and 10 weeks of treatment of <i>Pkd1</i>^–/– or wt mice. <i>n</i> = 6–8 mice. * indicates comparisons to <i>Pkd1</i>^–/–, vehicle-treated mice: *, <i>P</i>≤0.05. All data were graphed as mean ± standard error of the mean (SEM). No cysts were detected in any wt mice.</p

Elesclomol-induced ROS generation and cytoxicity in yeast is dependent on the presence of copper.

<p>(A) Chemical structure of the elesclomol-Cu complex. (B) The parental BY4743 yeast strain was grown in the presence of the indicated concentrations of elesclomol, preformed elesclomol-Cu, and/or copper for 21.5 h at 30°C. Absorbance at 600 nm was used to determine cell density. (C) Flow cytometric analysis measuring ROS in <i>S. cerevisiae</i>. ROS induction, as measured by Dihydrorhodamine 123 fluorescence, was only observed with preformed elesclomol-Cu complex at a concentration (500 nM) above the MIC but not below (100 nM), nor with free elesclomol at either concentration (<i>upper panel</i>). The addition of supplementary copper (via CuCl₂) to elesclomol was sufficient to induce ROS, again only at the higher concentration (<i>lower panel</i>). (D) Elesclomol is cidal to yeast cells within an hour of treatment. Logarithmically growing cells were incubated with the indicated doses of elesclomol for 1, 2 or 4 h and then plated onto media without elesclomol. 5 µM and 22.5 µM elesclomol rendered cells unviable within 1 h, and lower doses (1.25 µM) killed cells within 4 h.</p

Sensitivity of <i>S. cerevisiae</i> mutant strains to elesclomol-Cu.

<p>(A) A genome-wide readout of heterozygous strain sensitivity. In the plot, the X-axis orders all genes by their systematic name (hence, chromosome position) while the Y-axis is a measure of the ‘fitness’ of the strain deleted for the indicated gene grown in sub-lethal doses of elesclomol-Cu. The value on the Y-axis corresponds to −log₂ (ratio of normalized strain signal in treatment to DMSO control). Hence, the zero line represents equivalent growth in both conditions, while each unit above the line represents a 2-fold reduction in strain fitness. (B) Genome-wide profile of homozygous strain sensitivity. The data are presented as in <i>(A)</i>. Among the 150 most sensitive strains, the 137 having mitochondrial roles are highlighted color-coded: red (7 genes, mitochondrial genome maintenance), dark brown (8 genes, metal ion homeostasis), bright green (26 genes, mitochondrial localization), aqua (4 genes, mitochondrial, uncharacterized), blue (36 genes, ox-phos and respiration) mauve (5 genes, mitochondrial splicing), light gray (9 genes, response to stress) dark brown (26 genes, mitochondrial translation), dark gray (7 genes, mitochondrial import/export) and dark green (9 genes, mitochondrial tRNA).</p

Biological processes and protein complexes associated with sensitivity to elesclomol.

<p>(A) Each node represents a significantly enriched biological process/protein complex in the elesclomol chemogenomic profile as determined by GSEA. The size of a node corresponds to the number of genes annotated to the functional category. The width of an edge corresponds to the level of gene overlap between two interconnected categories (i.e. gene sets). Edges are not shown where the overlap coefficient is less than 0.5 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029798#s4" target="_blank">Materials and Methods</a>). The color of a node shows the cluster membership where clustering is based on the level of overlap between categories. (B) Each bar plot corresponds to the cluster indicated by its border color, and shows the individual sensitivity scores (X-axis) of the genes that contributed to the functional enrichments of the cluster. For clusters with more than 10 genes contributing to the enrichments, only the top 10 associated with the most categories are shown. The percentage of times the particular genes occur in that gene set is indicated by a color key, with black indicating the gene is present every time that function appears enriched, graded to white for those genes in the leading edge that appear less frequently within the gene set.</p

Combinations of elesclomol-Cu and ETC complex inhibitors in melanoma cells.

<p>(A) Single agent viability assays in Hs249T human melanoma cells using graded concentrations of elesclomol-Cu, antimycin A or rotenone for 72 h. The IC₅₀ values obtained were 11 nM, 240 nM and 77 nM, respectively. Combination treatment of elesclomol-Cu with antimycin A (B) or rotenone (C) at IC₂₀ or IC₅₀ doses resulted in significantly enhanced cytotoxicity, showing that direct modulation of the ETC and mitochondrial respiration in mammalian cells enhances the cellular activity of elesclomol.</p