E2 Neutralization Conditioned medium was incubated with 10 g/ml of 2B5 anti-PGE2 antibody for 30 minutes at room temperature prior to treating the HLFs. Statistics Statistical analysis was conducted 
using either one-way ANOVA with Tukey post-test or Student’s t-test using Prism software version 5. P values are listed in the figure legends. Results were considered significant when p<0.05. All raw data for the figures shown in this manuscript is contained in an online data supplement. 5 / 19 Epithelium Inhibits Myofibroblast Differentiation Results Human lung Indirubin-3′-oxime manufacturer epithelial cells inhibit TGF- induced -SMA expression in human lung fibroblasts To determine whether lung epithelial cells are capable of exerting anti-fibrotic effects on fibroblasts, we designed a co-culture system in which normal primary human lung fibroblasts are co-cultured with primary human small airway epithelial cells separated by permeable culture inserts. HLFs were treated with or without TGF-1 in the presence or absence of epithelial cells for 72 hours. Fibroblast lysates were assessed for expression of -smooth muscle actin, a marker of myofibroblast differentiation, by western blot. HLFs stimulated with TGF-1 differentiated to myofibroblasts and expressed -SMA, as we and others have reported. However, when co-cultured with SAECs, myofibroblast differentiation after TGF-1 stimulation was almost completely inhibited. In addition to reducing -SMA expression, co-culture with SAECs significantly reduced soluble collagen expression by TGF- treated HLFs compared to HLFs that were cultured alone. The ability of SAECs to inhibit myofibroblast differentiation was a consistent property of SAEC strains from multiple donors. Because it is currently unknown which PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723701 type of lung epithelial cells may be most important in IPF, we also examined the effect of freshly isolated alveolar epithelial cells. AECs also potently inhibited TGF- induced myofibroblast differentiation of human lung fibroblasts. SAECs inhibit TGF- induced -SMA expression in both normal and fibrotic human lung fibroblasts The ability of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19722795 SAECs to suppress myofibroblast differentiation was also a consistent property of multiple lung fibroblast strains. We evaluated primary HLFs from 2 additional non-fibrotic donors and from 3 IPF patients. Interestingly, SAECs were potently effective at inhibiting TGF- stimulated myofibroblast differentiation in both normal and fibrotic HLFs. Epithelial cells inhibit TGF- induced -SMA expression in fibroblasts irrespective of their tissue origin Epithelial cells and fibroblasts are contiguous in many injury and wound healing situations. Given our results that primary lung epithelial cells are capable of inhibiting TGF- induced -SMA expression in both normal and fibrotic lung fibroblasts, we examined whether epithelial cells were capable of protecting fibroblasts from pro-scarring insult irrespective of their tissue origin. Graves’ ophthalmopathy is an autoimmune disease in which orbital fibroblasts proliferate and differentiate to myofibroblasts in response to infiltrating T cells. Keloid scars represent a fibroproliferative disorder characterized by abnormal wound healing and high levels of -SMA and collagen expression. Using the same co-culture system, we cultured Graves’ orbital and keloid fibroblasts with SAECs. Interestingly, SAECs inhibited TGF- stimulated -SMA expression in both Graves’ orbital fibroblasts and keloid fibroblasts.. Next, we also wanted to determine whether epithelial