APECs was induced only with the combination of Ifn and higher 8 / 28 Ifn and Nos2 Regulate Functions of APECs Fig 2. Modulation of expression of different cell surface proteins on APECs in 181223-80-3 web response to Ifn. Kinetic analysis of cell surface amounts of P-Selectin, MHC class II, Icam1, Lfa1, E-Selectin and CD11b upon Ifn treatment of APECs. Fluorescence microscopic images acquired at 63X with a scale of 10 m of E-Selectin and CD11b on the cell surface of APECs treated with 25 U/ml of Ifn for 36 h. The data is represented as mean S.E from two independent experiments. doi:10.1371/journal.pone.0128301.g002 amounts of DETA/NO. Overall, these results clearly demonstrate that the combination of Ifn and NO derived signals is required for aggregation of APECs. Nos2 regulates the amounts of cell surface CD11b To investigate the roles of Nos2 in regulation of cell surface molecules by Ifn, further studies were performed. The induction of MHC II and repression of Lfa1 by Ifn were Nos2 independent. Interestingly, the lack of Nos2 led to increased basal expression of CD11b as well as E-Selectin. However, Ifn induced repression of E-Selectin and CD11b in C57BL/6 as well as Nos2-/- APECs to a similar extent. Further, fluorescence microscopic analysis revealed that lack of Nos2 led to diffused clustering and patchy cell surface localization of CD11b and E-Selectin, which was not 
observed on C57BL/6 APECs treated with Ifn. Most likely, Nos2 mediates APECs aggregation, in part, by regulating the expression and cell surface localization of some cell surface molecules, e.g. CD11b, in response to Ifn. Lack of Nos2 impacts morphology of APECs We observed that treatment of C57BL/6 APECs with LNMA or Nos2-/APECs, in the presence of Ifn led to a flattened cellular morphology. To understand the finer details, ESEM was performed. APECs from C57BL/6 mice appeared mostly spherical and formed stable aggregates in response to Ifn. On the other hand, the lack of Nos2 resulted in a highly flattened cell morphology and there was an absence of aggregate formation upon 9 / 28 Ifn and Nos2 Regulate Functions of APECs Fig 3. CD11b is required for aggregation of APECs in response to Ifn. The relative amounts of E-Selectin and CD11b on APECs upon treatment with Ifn in the absence or presence of indicated amounts of Reopro or siRNA to Cd11b was measured after 36 h. The amount of nitrite and cell aggregates upon 25 U/ml of Ifn treatment in the absence or presence of indicated doses of Reopro or 200 nM siRNA to Cd11b is shown. The data is represented as mean S.E from three independent experiments and control refers to untreated cells alone. The significance with respect to untreated controls and Ifn treated C57BL/6 APECs controls are represented as and respectively. doi:10.1371/journal.pone.0128301.g003 Ifn addition. Importantly, these alterations in morphology and aggregation upon Ifn addition in Nos2-/- APECs could be functionally reversed by adding SNAP exogenously, demonstrating NO dependence. These PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19697345 data highlight the requirement of Nos2 derived NO in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698359 conjunction with other Ifn signals in mediating aggregate formation as well as maintenance of morphology in APECs. Absence of Nos2 does not skew macrophage polarization signals in response to Ifn Ifn in known to polarize macrophages to the M1 phenotype and the induction of Nos2 is an important marker. Therefore, we investigated whether the effects observed in terms of morphology, motility and aggregation in Nos2-/- APECs were due
