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Additional file 3. Supplementary Materials and Methods.

Additional file 2: Supplementary Figure S1. Effects of C28 on microtubule organization in BC and non-transformed breast cell lines and schematic model depicting the proposed mechanism of action of C28 inhibitor. (a) MCF7, T47D, MDA-MB-231 and MCF12A cells were treated with 10 μM of C28 for 48 h. Cells were fixed and stained with anti-α-tubulin antibody (green) while the nuclear DNA was stained by DAPI (blue). Representative confocal microscopy images of mitotic phase cells are shown. Scale bar, 5 μm. (b) C28 binds to LMTK3 promoting its proteasome-mediated degradation. Downregulation of LMTK3 leads to a decrease in NUSAP1 and downstream proteins CDK1 and phospho-β III tubulin (S172), resulting in cell cycle arrest, abnormal spindles and microtubules instability.

Introduction The last years were characterized by several studies proceeding to understand SMC heterogeneity. Animal models of rat or rabbit IT after endothelial lesion are presently the most studied models for atherosclerotic plaque formation and have been useful in order to understand several biological features of SMCs. The disadvantages of animal models are that there is no possibility to reproduce the same history of pathogenesis in animals as that in man. There is no perfect animal model that completely replicates all stages of human atherosclerosis. Howevere, identification of particular SMCs subpopulation in human arteries represents a difficult challenge for understandable reason, such us material availability and experiment standardization. The relevance of SMC heterogenity to human disease still remains to be demonstrated. Aim Aim of this study is to obtain primary culture of human vascular smooth muscle cells (SMCs) from both atherosclerotic and no-atherosclerotic carotid artery, to investigare human SMC heterogeneity. Moreover, the possibility to isolate o SMC subpopulations, from a single human artery, could be usefull to verify the hypothesis that distinct SMC subtypes are involved in atherosclerosis pathogenesis and progression. Materials, Methods and Results Thanks to a collaboration between our laboratory and the Vascular Surgery’s group of Sant’Anna Hospital(University of Ferrara) leading by Francesco Mascoli, we had the possibility to obtain samples of atherosclerotic and noatherosclerotic human carotid artery from patients undergone to carotid endarterectomy. From these specimens we proceeded with tissue explantation in order to obtain primary colture of vascular smooth muscle cells both from no-atherosclerotic and atherosclerotic carotid artery. Cells obtained by tissue explantation were tested by immunoflurescences staining in order to verify the presence of alfa smooth muscle actin, a specific marker of vascular smooth muscle cells. Once we verified the nature of our cells through the presence of alfa smooth muscle actin we have tried to divide the atherosclerotic artery in media and plaque and than proceeding tissue explatation only with media or only with plaque or with complete lesion. We have found out that only from the complete lesion (media +plaque) we have obtained cellular outgrowth, while media and plaque alone has not gave cells at all. From these results we were started to understand which tissue, between media and plaque was the proliferative ones. In order to obtain this data we have proceeded with an experiment of cocolture using transwell insert, wich allows media and plaque to stay phisically separeted but to share the same medium. After these experiments, we have found out that the proliferative tissue was the media but plaque presence was a “condicio sine qua non” for cellular outgrowth in atherosclerotic carotid artery. We have compared cells coming from non atherosclerotic media with the ones coming from atherosclerotic artery: early, we have found out the morphological differences: cells from non atherosclerotic media was large and grew up in a monolayer even at confluence, while cells from atherosclerotic media was small and elongated and, at confluence, grew up forming a multilayer net. We have analyzed the cells also using immunofluorescences finding out that large cells(coming from no-atherosclerotic artery), had much alfa smooth muscle actin and did not shown Desmin, or S100A4(a marker of atheroma prone cells in porcine model of atherosclerosis), but show SMMHC. Small cells (coming from atherosclerotic artery), instead, had less alfa smooth muscle actin compared to large cells, and did not shown desmin and SMMHC but show an high production of S100A4. We also have tried to add plaque in tissue explantation of no-atherosclerotique media and the outgrownig from these experiments was small and by immunofluorescence shown the presences of S100A4. These are only preliminary data. The presence of the mRNA of these markers was analyzed by using RT-PCR. For further information about the differences between these two celluar populations we led a microarray experiment using biological material coming from three patiences. We discovered that there are 375 genes modulated in the two populations; these genes are involved in several biological pathways, in particular inflammation and cellular development. In conclusion we found that there are two kind of cellular populations in human artery with atherosclerotic lesion, they have some important differences that could explain why cells change phenotype and migrate in to the intima. Cells from atherosclerotic artery are strictly dependent of plaque presences that act on on them as a magnet. We identify a group of genes that can help us to understand what are the steps of phenotypical modulation. Presence of plaque nearby no-atherosclerotic artery can switch the cells from a large phenotype to a Small Phenotype (this preliminary data is confirmed by the presence in cells, outgrowed from no-atherosclerotic artery in presece of plaque, of S100A4)

Understanding how an epigenetic regulator drives different cellular responses can be a tricky task. Very often, their activities are modulated by large multiprotein complexes, the composition of which is context- and time-dependent. As a consequence, experiments aimed to unveil the functions of an epigenetic regulator can provide different outcomes and conclusions, depending on the circumstances. HDAC9 (histone deacetylase), an epigenetic regulator that influences different differentiating and adaptive responses, makes no exception. Since its discovery, different phenotypes and/or dysfunctions have been observed after the artificial manipulation of its expression. The cells and the microenvironment use multiple strategies to control and monitor HDAC9 activities. To date, some of the genes under HDAC9 control have been identified. However, the exact mechanisms through which HDAC9 can achieve all the different tasks so far described, remain mysterious. Whether it can assemble into different multiprotein complexes and how the cells modulate these complexes is not clearly defined. In summary, despite several cellular responses are known to be affected by HDAC9, many aspects of its network of interactions still remain to be defined.