Plk1 colocalizes with NPHP1 at the base of the cilium.

<p><b>A</b> Ciliated hTERT-RPE1 (human retinal pigmented epithelial cells and HK2 human kidney cells were stained with antibody to Plk1 (green), acetylated α-tubulin (orange), and γ-tubulin (red), and treated with DAPI to visualize DNA (blue). The scale bar represents 5 µm. <b>B</b> Ciliated hTERT-RPE1 cells and HK2 cells were stained with antibody to acetylated α-tubulin (orange), γ-tubulin (red), to NPHP1 or Plk1 as indicated (green), and with DAPI to visualize DNA (blue). The third row shows merged signals from staining with antibody to Plk1 (orange), NPHP1 (green) acetylated α-tubulin (orange), γ-tubulin (red) and DAPI was used to visualize DNA (blue). The scale bar represents 5 µm.</p

Plk1 associates with NPHP1.

<p><b>A</b> Western blot of immunoprecipitates (IP) or lysates (Lys) from HEK293T cells co-transfected with plasmids expressing V5-tagged NPHP1 and Flag-tagged Plk1 or negative control protein (Eps1–225 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038838#pone.0038838-Habbig1" target="_blank">[13]</a>). β-actin was assessed as a loading control. <b>B</b> Western blot of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells co-transfected with plasmids expressing Myc-tagged Plk1 and Flag-tagged NPHP1 or empty Flag vector. <b>C</b> Western blot of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells transfected with plasmid expressing Flag-tagged NPHP1 or the negative control protein (Eps1–225 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038838#pone.0038838-Habbig1" target="_blank">[13]</a>). Endogenous Plk1 was detected using a specific antibody against Plk1. <b>D</b> A panel of Flag-tagged NPHP1 derivatives, including truncations, internal deletions and a T87A mutant, was analyzed by co-immunoprecipitation with Myc-tagged Plk1. <b>E</b> Western analysis of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells co-transfected with plasmids expressing Myc-tagged Plk1 and Flag-tagged NPHP1 constructs as indicated, or the Flag-tagged control protein (Eps1–225). * indicates immunoglobulin heavy chain.</p

Plk1 directly phosphorylates NPHP1 in vitro.

<p><b>A</b> Alignment of NPHP1 protein sequences from multiple species indicates a conserved candidate Plk1 motif at position T87. <b>B</b> An <i>in vitro</i> kinase assay performed with active Plk1 and recombinant His-fused NPHP1 protein indicates phosphorylation within the NPHP1 N-terminal 205 amino acids. CB, Coomassie Blue.</p

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

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

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