FKBPL is present in various cell compartments and regulates cell migration and tumour vasculature.

<p>(<b>A</b>) Representative blot demonstrating that FKBPL is present predominantly in the cytosol and membrane compartments of both HMEC-1 (H) and MDA-MB-231 (M) cells and in the nuclear fraction of MDA-MB-231 cells<b>.</b> Protein extracts from each subcellular compartment probed with specific compartmental markers, vimentin, calpain and histone-H1 were used as loading controls. (<b>B</b>) Representative confocal images (60x) of MDA-MB-231 and HMEC-1 cells fixed, permeabilised and stained with DAPI (blue) and with anti-AD-01 primary antibody and Alexa-488 tagged secondary antibody demonstrating vesicular staining for FKBPL (green); n = 3. (<b>C</b>) Anti-AD-01 antibody targets the active domain of FKBPL and accelerates HMEC-1 cell migration in comparison to cells treated with an isotype control. Data points show means ± SEM; n = 3 (<b>D</b>) FKBPL knockdown with siRNA accelerated migration of HMEC-1 cells in comparison to un-transfected and NT-siRNA-transfected cells. Data points show means ± SEM; n = 3. Cell migration was assessed using scratch wound assay. Wound size is normalised to that of T₀. p-value was determined using two-way ANOVA. (<b>E</b>) Intravital microscopy images (20x) representing disruption of tumour vasculature <i>in vivo</i> in FKBPL-overexpressing MDA-MB-231 xenografts in comparison to those derived from parental MDA-MB-231 cells. Tumours (21 days) were imaged using Epi-fluoresence microscopy following injection of mice with FITC-Dextran. Quantification of vessel dynamics was carried out on 3D images using ImageJ software. n = 5 mice per treatment group (p-value was determined using two-tailed T –test).</p

FKBPL and its peptide derivative, AD-01, bind CD44.

<p>(<b>A</b>) Representative western blot showing FKBPL co-immunoprecipitated with CD44 in HMEC-1 cells; immuno-blotted with anti-CD44 antibody; n = 3; Cdc42 was used as a positive control for the CD44 interaction and rabbit and murine IgGs were used as negative controls. (<b>B</b>) Schematic diagram of the Biacore assay using AD-01 immobilised on CM5 chip surface. Binding of anti-AD-01 antibody to AD-01-CM5 surface was inhibited by AD-01 in solution in a dose dependent manner, with excellent sensitivity in lower concentration range of peptide; 1–500 nM. Scrambled AD-01, used as a negative control, did not demonstrate any binding to anti-AD-01 up to 200 µM. Competition of the anti-AD-01 antibody interaction with its cellular partner/s results in increased binding of anti-AD-01 antibody on chip surface. (<b>C</b>) Representative graph demonstrating that AD-01 specifically binds to CD44 immunoprecipitated from MDA-MB-231 cells using the assay described. CD44 was immuno-purified from cell lysate and analysed using Biacore Q. Isotype control mIgG antibody was used as control. Bar charts show the relative binding of anti-AD-01 antibody in presence of AD-01, calculated as the percentage of the maximum resonance binding units in the presence of 0.001 and 0.01 µM AD-01. Data points show means ± SEM of 5 independent experiments (p-value was determined by one way ANOVA). (<b>D</b>) No competition of anti-AD-01 antibody binding to immobilised AD-01 was obtained in the presence of various concentrations of rEGFR indicating a specificity of AD-01-CD44 interaction.</p

AD-01 and FKBPL mediate cytoskeletal changes

<p><b>and AD-01 disrupts the RhoA-Rac1 dynamics.</b> (<b>A</b>) Confocal images (60x) representing the changes in phalloidin/F-actin dynamics in MDA-MB-231 upon wounding and treatment with AD-01 (10⁻⁹M) for 24 h; n = 3. Treated monolayers were fixed and stained with TRITC-phallodin (red) and DAPI (blue). Intense actin staining was accompanied by the loss in cell direction and communication. (<b>B</b>) Images (40x) representing disruption in tubulin distribution in HMEC-1 cell monolayers, wounded and treated with rFKBPL 750 ng/ml for 5 h. Fixed monolayers were stained for tubulin, followed by FITC conjugated secondary antibody (green) and nucleus with PI (red). (<b>C</b>) RhoA expression was increased after wounding and treatment with AD-01 for 24 h, resulting in a concomitant increase in the downstream actin binding proteins vinculin and profilin. Cells monolayers were treated with AD-01 (± wounding) for 24 h and total cell lysates were subjected to immuno blotting as indicated. (<b>D</b>) Treatment with AD-01 (10⁻⁹M) for 10/60 min inhibited fMLP induction (30 sec) of GTP-Rac-1 in HMEC-1 cells. Cell lysates of treated monolayers were subjected to Rac GTPase pull down assay; n = 3. (<b>E</b>) Representative western blots and quantitative densitometric analysis demonstrating an inhibition of cofilin phosphorylation after treatment with AD-01. HA treatment up-regulated cofilin phosphorylation maintaining its inactive state. HMEC-1 cell monolayers were treated with AD-01 for 3 h or HA for 10 min, and the extracted membrane fractions were subjected to immunoblotting with cofilin/p-cofilin; n = 3.</p

Heterogeneity of rucaparib activity in arteries isolated from patients having undergone nephrectomy.

<p>Panel A; concentration-response curves that were generated. Tissues were constricted using 10 μM PE, before being treated with PE and the relevant concentration of rucaparib. Panels B1 and B2; responses of two separate arterial sections from a single donor; panel B1 details the relaxant activity when a single tissue segment was constricted using 10 μM PE before being relaxed using 100 μM rucaparib; panel B2 details the slight inhibition of spontaneous oscillation of a single tissue segment that was observed following treatment with 100 μM rucaparib (the tissue contracted spontaneously, so no PE constriction was performed). Points/bars in most cases represent mean of two parallel experiments. Error bars represent SEM. Panels B1 and B2 error bars, as only a single observation was made per tissue section.</p

Effects of rucaparib on tumor vessel perfusion may be dependent on PARP.

<p>Panels A and B; B16 tumours were established in PARP WT or KO female mice. The extent of vessel ‘mismatch’ following the administration of the perfusion markers Hoechst 33342 and carbocyanin was reduced by rucaparib (1 mg/kg) in tumors established in WT but not PARP-1^-/- mice. Panel C; fold change in intratumoral fluorescence above that seen following initial plateau (20 min) in dorsal window chambers implanted with B16 tumors in WT and PARP-1^-/- mice and treated with 1 mg/kg rucaparib. Panel D; representative real time analysis of the accumulation of BSA-647 (administered via iv injection at time 0) in B16 tumors established in dorsal window chambers in PARP WT (closed symbols) and PARP-1^-/- (open symbols) mice. Arrow indicates the administration of rucaparib (10 mg/kg). NS—p >0.05, *p<0.05, **p<0.01 as compared with relevant control. N = 3 mice per condition.</p

Rucaparib may act at multiple P2 receptor subtypes.

<p>Panels A and B; blockade of specific P2 receptor subtypes P2X₄, P2X₇ and P2Y₁ in tail artery (A) and aorta (B) models failed to categorically identify the receptor subtype at which rucaparib elicits its dilatory effect. ^Δ p<0.05 as compared with the degree of dilation achieved by rucaparib in the absence of P2 antagonism. Panel C; summary of the duration of perfusion of tail artery segments necessary for relaxation plateau to be reached. Although the absolute degree of relaxation achieved was similar in the absence and presence of specific P2 receptor antagonism, the time taken for relaxation to complete was prolonged in all cases tested in tail artery. ***p<0.001 compared to time taken for relaxation plateau in the absence of P2 antagonism. Panel D; representative trace of rucaparib-induced relaxation when the P2Y₁ receptor was antagonised using MRS-2179 (top), and rucaparib-induced relaxation (bottom). Bars represent mean of at least three independent experiments. Error bars represent SEM. Arteries from at least three rats were used per test.</p

P2 receptor blockade abrogates rucaparib-evoked vasodilation.

<p>Panel A; broad-spectrum antagonism of P2 receptors using suramin (inverted open triangles) abrogated rucaparib-evoked relaxation in rat tail artery (left) and aorta (right). Panel B; suramin itself was without vasoactivity, demonstrable by its failure to alter the tone of sub-maximally constricted vessels. The shaded region represents the degree of constriction evoked using 10 μM PE alone. Panel C; suramin has little effect on vasodilation elicited by nicotinamide and none on vasodilation elicited by ML-9, but inhibits that elicited by rucaparib in tail artery and aorta. **p<0.01, ***p<0.001 as compared with dilation achieved in the absence of suramin. Arteries from at least three rats were used per test.</p

Rucaparib-mediated vasodilation of rat vascular tissue may be partially dependent on myosin light chain kinase.

<p>Panel A; rucaparib (closed squares), nicotinamide (open squares) and ML-9 (open triangles) inhibit smooth muscle contraction in PE-constricted rat tail artery. Artery sections were constricted using 10 μM PE before perfusion with a solution containing 10 μM PE plus the relevant concentration of drug. Panel B; rucaparib, nicotinamide and ML-9 inhibit smooth muscle contraction in PE-constricted rat aorta. Panel C; inhibition of arterial smooth muscle contraction by rucaparib is dependent on a mechanism in addition to MLCK inhibition. Constricted vessel segments were relaxed to the maximal degree achievable with ML-9, before being challenged with a relaxing cocktail of ML-9 plus rucaparib. The histograms illustrate the additive effects that were observed in the cases of both tail artery (left) and aorta (right). ** p<0.01, *** p<0.001 versus relaxation evoked by rucaparib alone; ^ΔΔ p<0.01 versus ML-9 alone. Bars represent mean of at least three independent experiments. Arteries from at least three rats were used per test. Error bars represent SEM. Panel D; rucaparib (closed squares) inhibits MLCK activity with ten times the potency of ML-9 (open triangles). Kinase activity was analyzed using the Millipore IC₅₀<i>Profiler</i> Express service. Points represent results of duplicate experiments. Error bars represent SEM.</p

Summary of rucaparib activity in PE-constricted human tumor-associated vasculature.

<p>^aData presented represents the magnitude of vessel relaxation in response to the top concentration of rucaparib tested, except A5, where the second highest concentration is summarised.</p><p>^bVessel section B1 was constricted with PE as the ‘A’ vessels; B2 contracted spontaneously, so is not summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118187#pone.0118187.t001" target="_blank">Table 1</a>.</p><p>Summary of rucaparib activity in PE-constricted human tumor-associated vasculature.</p