DMSO or the different compounds were added 2 h after thymidine release. Figure S2: L-Ornithine Effects of Tripolin A and MLN8237 on centrosome organization. (A) Representative immunofluorescence images of HeLa cells in metaphase, treated with solvent control (DMSO) or 20 M Tripolin A for 24 h. In the merged images TPX2 is pseudocolored red, -tubulin green, DNA blue. (Scale bars 5 m). (B) Images of mitotic HeLa cells treated with solvent control (DMSO) or 100 nM MLN8237 for 5 h and 24 h. In the merged images Aurora A is pseudocolored red, pericentrin green, DNA blue. (Scale bar 5 m). (C) Graphs showing the percentage of mitotic cells with fragmented centrosomes (up), or acentrosomal poles (down) in control mitotic cells (DMSO) and mitotic cells treated with MLN8237 for 5 h and 24 h. (n?=?150 cells for each group, from three L-Ornithine independent experiments).(TIF) pone.0058485.s002.tif (1.1M) GUID:?AB8AE5BC-E923-4C7B-933A-409BD9FED3E8 Figure S3: Aurora A depletion by siRNA does not affect MT binding of HURP. Fluorescence intensity (arbitrary units) of HURP bound on spindle MTs was quantified in control and Aurora A depleted metaphase cells (n20 cells for each group, from at least two independent experiments). ***: p<0.001; ns: p>0.05; (Mann-Whitney test, two-tailed). Error bars represent SEM.(TIF) pone.0058485.s003.tif (62K) GUID:?FF29C5DE-27B3-4AC1-B047-C16A47B6D6E0 Figure S4: single cell and studies to demonstrate the biological action of Tripolin A, a non-ATP competitive inhibitor. Tripolin A reduced the localization of pAurora A on PJS spindle microtubules (MTs), affected centrosome integrity, spindle formation and length, as well as MT dynamics in interphase, consistent with Aurora A inhibition by RNAi or other specific inhibitors, such as MLN8054 or MLN8237. Interestingly, Tripolin A affected the gradient distribution towards the chromosomes, but not the MT binding of HURP (Hepatoma Up-Regulated Protein), a MT-associated protein (MAP) and substrate of the Aurora A kinase. Therefore Tripolin A reveals a new way of regulating mitotic MT stabilizers through Aurora A phosphorylation. Tripolin A is predicted to bind Aurora A similarly but not identical to MLN8054, therefore it could be used to dissect pathways orchestrated by Aurora kinases as well as a scaffold for further inhibitor development. Introduction Temporal and spatial coordination of the process of mitosis and cytokinesis is a prerequisite for accurate and equal segregation of genomic and cytosolic material into two daughter cells. Among the network of regulatory proteins, Aurora kinases are of particular importance. In terms of enzymatic activity, Aurora kinases belong to the Ser/Thr kinase family and they comprise of two domains: a regulatory domain at the NH2-terminus and a catalytic domain at the COOH-terminus. Auroras share a great degree of homology in their catalytic domain, whereas differ in their NH2-terminal domain. The mammalian orthologs are at least three: Aurora A, Aurora B and Aurora C . By means of phosphorylating different substrates, including TPX2 , Ajuba , TACC3 , , Eg5  and HURP ,  among others, Aurora A is implicated in diverse cell cycle events: centrosome maturation and separation, mitotic entry, bipolar spindle assembly, chromosome alignment, spindle checkpoint and cytokinesis. TPX2 is not merely a substrate but also the best-studied activator of Aurora A, required for Aurora A localization to spindles . Moreover, Aurora A regulates the mitotic spindle apparatus as part of a multi-protein complex along with the kinesin Eg5 and three MAPs; TPX2, XMAP215 L-Ornithine and HURP . HURP is a MT stabilizer with distinct features since it localizes mainly to kinetochore MTs (kt-MTs) of the mitotic spindle ,  and induces a unique MT conformation kinase assays was determined. Two L-Ornithine compounds (OXVW5 and OXVW25) showing an inhibition greater than 70%, at a concentration of 10 M were further investigated and hereafter referred to as Tripolin A and Tripolin B, respectively (Figure 1A). Open in a separate window Figure 1 Tripolins inhibit Aurora kinase activity kinase assay. (C) Differential Scanning.