Notably, some known susceptibility genes of FS were also identified in the DEGs

ortical F-Actin. In case of -Tubulin, it was found evenly spread in the cytoplasm. Importantly, in the absence of Nos2, addition of Ifn led to displacement of the cortical Actin and Tubulin network. This defect in Nos2-/- APECs was not reversed by the exogenous addition of SNAP alone. However, the combination of SNAP with Ifn led to restoration of the cortical arrangement of Actin and Tubulin in Nos2-/- APECs. This aspect of regulation of cortical stability of network of Actin and Tubulin by Nos2 in APECs, only in the presence of Ifn has been quantified and represented in Fig. E in S1 File. Actin and Tubulin stability contribute to motility, morphology and aggregation of APECs To “directly” assess the roles of stabilization of Actin and Tubulin during the aggregation of APECs, studies were performed with Cyt D, a potent Actin depolymerizing compound, and Col, a potent microtubule depolymerizing agent. First, Cyt D and Col treatment led to Actin depolymerization and shrinkage of the Tubulin network as revealed by their retraction from the cell membrane in C57BL/6 APECs. These inhibitors did not affect the Ifn induced nitrite production by APECs, confirming our observations that the Actin and 12 / 28 Ifn and Nos2 Regulate Functions of APECs Fig 6. Nos2 regulates the morphology of APECs treated with Ifn. Addition of either of these inhibitors, in a dose dependent manner, led to significant reduction in the formation of aggregates in response to Ifn. Interestingly, addition of Cyt D reduced, whereas Col addition increased, basal amounts of E-Selectin and CD11b PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698015 levels on the cell surface without affecting their fold repression upon Ifn addition. Live cell imaging analysis revealed that addition of Cyt D, but not Col, reduced basal motility of APECs. However, addition of Col, but not Cyt D, led to flattened morphology of APECs, similar to those seen in Nos2-/- APECs treated with Ifn. Most likely, Actin stabilization contributed to the motility, whereas Tubulin stabilization was important for the maintenance of morphology in APECs. Both Actin and Tubulin were regulated by Nos2 generated NO in response to Ifn treatment in these cells. To address the roles of the extent of NO in affecting motility of APECs, experiments with low and high dose of SNAP were performed. Live cell imaging analysis revealed that addition of Ifn or CytD, but not Col, reduced the basal motility and velocity of C57BL/6 APECs. Also, the lack of Nos2 reduced basal motility and velocity when compared to C57BL/6 APECs. Interestingly, addition of low dose of SNAP to Nos2-/- APECs significantly increased basal motility and velocity to that of C57BL/6 APECs controls. Importantly, addition of higher dose of SNAP ONX-0914 chemical information lowered basal motility and velocity in Nos2-/APECs. It is likely that there are thresholds for the effects of NO on Actin and Tubulin that may be important in determining the extent of motility of APECs. 13 / 28 Ifn PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698988 and Nos2 Regulate Functions of APECs Fig 7. The cortical stability of cytoskeleton elements, Actin and Tubulin, is regulated in a Nos2 dependent manner upon Ifn treatment. Fluorescence microscopic images, with the scale bar of 10 m, of F-Actin and -Tubulin in APECs from C57BL/6 mice and Nos2-/- mice treated without or with 25 U/ml of Ifn for 36 h. In addition, Nos2-/- APECs were treated with 100 M of SNAP in the absence or presence of Ifn. The white arrows indicate cortical arrangement of F-Actin and -Tubulin. doi:10.1371/journal.pone.0128301.g007 14 / 28 Ifn