Verslips, CSF-1 deprived then re-stimulated with CSF-1 for 5 minutes. Cells were fixed and stained for F-actin and cell area and cell elongation analysis was conducted using ImageJ software. Representative of three independent experiments with .30 cells measured for each experiment. Where blue bars represent CSF-1 starved and red bars represent CSF-1 stimulated cells ** = p,0.005. doi:10.1371/journal.pone.0054869.gNox22/2 Cells did not Exhibit a Chemotactic Response Towards CSF-BMM are known to have a chemotactic response to CSF-1 [17], and in a physiological context are likely to be responding to a gradient of chemoattractant rather than global stimulation. Thus we next challenged the WT and Nox2KO BMMs to chemotax towards a source of CSF-1 using the Dunn Met-Enkephalin chemical information Chemotaxis Chamber. Whilst WT BMM were able to efficiently chemotax towards CSF1, Nox2KO BMMs completely lost their chemotactic response (Figure 4). Loss of chemotaxis can sometimes be attributed to a reduction in cell speed and we did indeed find that there was a significant reduction in mean cell migration speed in the Nox2KO population (p,0.001) (Figure 4C). However, we found that cell persistence was also significantly (p,0.001) reduced in Nox2KO BMM (Figure 4D) as compared to WT suggesting that Nox2KO cells were unable to respond to the CSF-1 gradient. This would MedChemExpress AN-3199 suggest a more significant role for Nox2 in the directed migration of the BMMs compared to random migration.Nox22/2 Macrophages have an Attenuated Signalling Response to CSF-Given that we have detected changes in both cellular morphology, cell spreading and directed cell migration we reasoned that signalling downstream of CSF-1 may be altered in Nox2KO cells. CSF-1 is well known to stimulate both ERK [19] and Akt phosphorylation [20] in BMMs. Indeed, levels of ERK phosphorylation have been 
linked to cell spreading [19]. We found no difference in Akt phosphorylation downstream of CSF-1, however, Nox2KO BMMs have an attenuated phospho-ERK response (Figure 5). Where levels of ERK phosphorylation were significantly reduced following 15 mins of CSF-1 stimulation. Thus, Nox2KO BMM do have attenuated signalling downstream of CSF-1 stimulation.DiscussionThe coordination and synergy between the cytoskeletal dynamics at the leading edge, the strengthening of adhesion to the ECM and cellular contractility play a key role in the dynamics of cellular morphology and migration [21]. Redox signalling hasbeen shown to be influential in this process at many different stages. In this paper Nox2 has been shown to play a role in regulating cellular morphology, random cellular motion and also to be critical in directed cellular migration, speed and chemotaxis. A key finding in this paper was that Nox2 in BMM was found to be important in random cellular motion and necessary in directed cellular motion. WT and Nox2KO BMMs are morphologically different and this is reflected in the difference in mean spread area of these cells and the elongated shape of Nox2KO BMMs. However, both populations were able to respond to CSF-1. Although, the Nox2KO BMMs tended towards a slower response in CSF-1 induced cell spreading. The loss of Nox2 did result in a significant reduction in the random motility of BMM as observed by the lower numbers of BMM migrating to the set horizon following CSF-1 stimulation. Also the Nox2KO BMM showed more intrinsic persistence in their random movement. Random motion allows cells to explore their environment. The increased intrin.Verslips, CSF-1 deprived then re-stimulated with CSF-1 for 5 minutes. Cells were fixed and stained for F-actin and cell area and cell elongation analysis was conducted using ImageJ software. Representative of three independent experiments with .30 cells measured for each experiment. Where blue bars represent CSF-1 starved and red bars represent CSF-1 stimulated cells ** = p,0.005. doi:10.1371/journal.pone.0054869.gNox22/2 Cells did not Exhibit a Chemotactic Response Towards CSF-BMM are known to have a chemotactic response to CSF-1 [17], and in a physiological context are likely to be responding to a gradient of chemoattractant rather than global stimulation. Thus we next challenged the WT and Nox2KO BMMs to chemotax towards a source of CSF-1 using the Dunn Chemotaxis Chamber. Whilst WT BMM were able to efficiently chemotax towards CSF1, Nox2KO BMMs completely lost their chemotactic response (Figure 4). Loss of chemotaxis can sometimes be attributed to a reduction in cell speed and we did indeed find that there was a significant reduction in mean cell migration speed in the Nox2KO population (p,0.001) (Figure 4C). However, we found that cell persistence was also significantly (p,0.001) reduced in Nox2KO BMM (Figure 4D) as compared to WT suggesting that Nox2KO cells were unable to respond to the CSF-1 gradient. This would suggest a more significant role for Nox2 in the directed migration of the BMMs compared to random migration.Nox22/2 Macrophages have an Attenuated Signalling Response to CSF-Given that we have detected changes in both cellular morphology, cell spreading and directed cell migration we reasoned that signalling downstream of CSF-1 may be altered in Nox2KO cells. CSF-1 is well known to stimulate both ERK [19] and Akt phosphorylation [20] in BMMs. Indeed, levels of ERK phosphorylation have been linked to cell spreading [19]. We found no difference in Akt phosphorylation downstream of CSF-1, however, Nox2KO BMMs have an attenuated phospho-ERK response (Figure 5). Where levels of ERK phosphorylation were significantly reduced following 15 mins of CSF-1 stimulation. Thus, Nox2KO BMM do have attenuated signalling downstream of CSF-1 stimulation.DiscussionThe coordination and synergy between the cytoskeletal dynamics at the leading edge, the strengthening of adhesion to the ECM and cellular contractility play a key role in the dynamics of cellular morphology and migration [21]. Redox signalling hasbeen shown to be influential in this process at many different stages. In this paper Nox2 has been shown to play a role in regulating cellular morphology, random cellular motion and also to be critical in directed cellular migration, speed and chemotaxis. A key finding in this paper was that Nox2 in BMM was found to be important in random cellular motion and necessary in directed cellular motion. WT and Nox2KO BMMs are morphologically different and this is reflected in the difference in mean spread area of these cells and the elongated shape of Nox2KO BMMs. However, both populations were able to respond to CSF-1. Although, the Nox2KO BMMs tended towards a slower response in CSF-1 induced cell spreading. The loss of Nox2 did result in a significant reduction in the random motility of BMM as observed by the lower numbers of BMM migrating to the set horizon following CSF-1 stimulation. Also the Nox2KO BMM showed 
more intrinsic persistence in their random movement. Random motion allows cells to explore their environment. The increased intrin.