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