erefore developed a cell culture model to explore the effects of type 1 IFNs on muscle cells. We found that type 1 IFNs impair myotube differentiation of both C2C12 mouse myoblasts and human myoblasts. These findings are consistent with other studies demonstrating toxic and siRNA Transfection For small interfering RNA experiments, C2C12 myoblasts were transfected 24 h after seeding, when cell density was approximately 3050%, with siRNA against ISG15 Western blots of ISG15 demonstrate successful silencing of the 15 kDa ISG15 protein with siISG15 treatment in IFN-b treated C2C12 cells. Note absence of 15 kDa bands at 48 h and 72 h, with partial return of protein expression at 96 h and 120 h. siISG15 also reduces ISG15 conjugates at all time points. Actin controls shown below. AZD-6482 Images demonstrate no improvement in myotube formation with ISG15 silencing at 72 h, and quantitative analysis shows ISG15 silencing with siISG15 results in no recovery of myotube area at 72 h, 96 h, and 120 h. doi:10.1371/journal.pone.0065362.g002 gen, Carlsbad, CA) at the final concentration of 30 nM per well. Four to six hours after transfection, cells were treated daily with two different doses of mouse IFN-a or -b; differentiation was induced 48 hours later. Measurement of Length, Diameter, and Area of Newly Formed Myotubes For quantitative morphological analysis, four micrographs were taken per well, at several time points, 48 h, 72 h, 96 h, and 120 h from the start of treatment) and for each, the length, diameter and area of the 10 visually largest myotubes were measured, using ImageJ 1.44 software. Statistical testing used nonparametric methods that do not assume Gaussian distributions of the data. ed and centrifuged for 10 min at 4uC, supernatants were collected and protein 10401570 contents were measured using BCA assay. Samples were diluted in Loading Buffer 4X and denatured for 5 min at 95uC. Equal amounts of proteins were electrophoresed on 
412% NuPage acrylamide gels, and transferred onto nitrocellulose membranes. Membranes were blocked in Tris buffered saline with 0.1% Tween 20 with 5% nonfat milk powder and incubated overnight in the same solution containing rabbit polyclonal anti-ISG15, at 4uC, followed by a 1 hr incubation at RT with secondary antibody. Immunoreactivity was detected by SuperSignal West Pico Maximum Sensitivity Substrate 5 min room temperature and exposed to film. Western Blotting Analysis Trypsinized cells were lysed in buffer containing 10 mM HEPES, 10 mM KCl, 1 mM EDTA, 0.1 nM EGTA, 10 mM DTT, 5 mM MgCl2 11325787 and Roche Complete Protease Inhibitor. Homogenates were sonicat- Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development and for the maintenance of homeostatic equilibrium in adult tissues. Stromal cells maintain control over epithelial cell proliferation, survival and response to wounds and could also play a collaborative role in pathological conditions such as cancer. The interactions between epithelium and mesenchyme are miR-155 Function in Fibroblast alpha, fibroblast growth factors, interleukin-1, monocyte chemoattractant protein-1 are produced at the sites of active fibrosis. These cytokines appear to be mostly expressed by activated inflammatory cells, such as macrophages and eosinophils. A balance of pro-fibrogenic and antifibrogenic forces generated by these networks of cytokines determines the outcome of lung injury and inflammation. In vitro studies of individual cytokines on
