and Nos2 Regulate Functions of APECs Fig 8. Actin and Tubulin stabilization contribute to Ifn induced aggregation of APECs. Fluorescence microscopic images acquired at a magnification of 63X with the scale bar representing 10 m of F-Actin and -Tubulin in C57BL/6 APECs treated with 10 M Cyt D and 1 g/ml of Col respectively for 6 h. Yellow and white arrows indicate cortical arrangement of F-Actin and -Tubulin respectively. Thin white arrows indicate the cell boundary while the fatter white arrows are used to highlight the shrinkage of the -Tubulin network upon Col treatment when compared to the untreated control C57BL/6 APECs. The amount of nitrite in the supernatant produced by APECs from C57BL/6 mice upon treatment with 25 U/ml of Ifn in the absence or presence indicated doses of inhibitors Cyt D and Col. The number of cell aggregates of APECs from C57BL/6 mice upon treatment with 25 U/ml of Ifn in the absence or presence of indicated doses of inhibitors Cyt D and Col. The amounts of E-Selectin and CD11b on the cell surface of C57BL/6 APECs treated with 25 U/ml of Ifn for 36 h, post 6 h of pretreatment without or with Cyt D and Col. The data is represented as mean S.E from three independent experiments. The velocity of APECs from 
C57BL/6 and Nos2-/- mice treated without or 15 / 28 Ifn and Nos2 Regulate Functions of APECs with 25 U/ml of Ifn between 1824 h of addition. The velocity of APECs from C57BL/6 pretreated for 6 h with Cyt D and Col before tracking them from 612 h post treatment. Also, the velocity of Nos2-/- APECs pretreated in the presence of 5 M of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698015 SNAP and 100 M of SNAP for 12 h and without or with 25 U/ml of Ifn before tracking cells 1824 h post Ifn addition. The velocity for each of the above mentioned conditions are represented as percentage velocity with respect to average velocity exhibited by untreated C57BL/6 APECs. The data is representative of two independent experiments. Significance is represented as when compared to untreated controls and # when compared to Ifn treated C57BL/6 APECs respectively. The significance with respect to untreated controls, Ifn treated C57BL/6 APECs controls. untreated Nos2-/- and SNAPlo alone Nos2-/- APECs controls are represented as ,,, and # respectively. doi:10.1371/PF-562271 custom synthesis journal.pone.0128301.g008 As macrophages are professional phagocytes, the possible contributions of Nos2, Actin and Tubulin to the phagocytic ability of APECs was evaluated using the internalization of latex beads. The phagocytic ability of C57BL/6 APECs was reduced with LNMA and CytD, but not Col, treatment. Also, loss of Nos2 decreased the phagocytic ability of APECs considerably. Further confirmatory experiments were performed using APECs from C57BL/6 and Nos2-/- mice that were infected with S. Typhimurium and their internalization, i.e entry into cells, was monitored after 2 h post infection. As seen in Fig. F in S1 File, the CFU recovered from Nos2-/- APECs were significantly reduced in comparison to their C57BL/6 counterparts. Overall, the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19697345 amounts of Nos2 generated NO regulate several functions of APECs: motility, phagocytosis, morphology and aggregation. APECs from S. Typhimurium infected mice aggregate in a Nos2 dependent manner It was important to address the biological relevance of Nos2-mediated modulation of functional responses in APECs. The approach utilized was to study the response of APECs from C57BL/6 and Nos2-/- mice post oral infection with S. Typhimurium. The amounts of cytokines, Tnf Interleukin 6 and I
