cells from other sources could inhibit myofibroblast differentiation of lung fibroblasts. We co-cultured lung fibroblasts with either primary human neonatal epidermal keratinocytes or the human lung alveolar epithelial cell line, A549, in the presence of TGF-. Epithelial cells from diverse origins suppressed myofibroblast differentiation. 6 / 19 Epithelium Inhibits Myofibroblast Differentiation Fig 2. SAECs inhibit TGF- induced -SMA protein expression in multiple normal and fibrotic human lung fibroblast strains. Two additional normal, and three additional IPF fibroblast strains from different donors were treated with or without 5ng/ml TGF- and co-cultured with SAECs for 72 hours. -SMA protein expression was analyzed by western blot. Representative blots are shown. Note that in Fig 2A, the indicated samples were resolved on the same gel, and intervening irrelevant lanes are not shown. Densitometry of n = 3 replicates per cell strain, normalized to untreated control. Data shown are mean SD. Epithelium Inhibits Myofibroblast Differentiation Fig 3. Epithelial cells inhibit TGF- induced -SMA protein expression in fibroblasts irrespective of their tissue origin. Multiple strains of human epithelial cells and fibroblasts were treated with or without 5ng/ml TGF and co-cultured for 72 hours. SAECs co-cultured with Graves’ orbital fibroblasts. SAECs PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723293 co-cultured with keloid fibroblasts. Human epidermal keratinocytes-neonatal co-cultured with HLFs. A549 cells cocultured with HLFs. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723429 -SMA protein expression was analyzed by western blot. Each lane represents a replicate culture. Representative blots are shown from at least two independent experiments per condition.Epithelium Inhibits Myofibroblast Differentiation Fig 4. SAECs inhibit collagen gel contraction and fibroblast migration. HLFs were seeded into collagen gels and floated in medium AZ-6102 containing 5ng/ml TGF-. SAECs were grown separately on Transwell inserts and added to the wells. After 72 hours gels were weighed and percent contraction was calculated. n = 34 per group. HLFs were grown on 6-well plates until they formed a confluent monolayer. A scratch wound was made on HLF monolayer, cells were washed with PBS and co-cultured with SAECs. HLF migration was tracked over time and imaged by phase contrast microscopy. The scratch assay was performed on 3 replicate cultures for each condition. Each culture was photographed at 3 locations, and the width of the scratch was determined at 3 positions in each photograph, and percentage of original width was calculated by measuring the width between the edges of the scratch wound in three distinctive areas of each image. Data shown are mean SEM.Human lung epithelial cells decrease collagen gel contraction and migration of human lung fibroblasts We also studied whether epithelial cells were capable of inhibiting other key pro-fibrotic effector functions exhibited by 
lung fibroblasts, specifically increased contractile ability and migration. Collagen gel contraction assays measure the ability of fibroblasts to organize and contract a collagen matrix in vitro. As predicted, TGF- increased contraction of collagen gels by the fibroblasts, but the fibroblasts that were co-cultured with SAECs failed to contract in response to TGF-. Next, we evaluated whether epithelial cells were capable of inhibiting fibroblast migration. Fibroblasts that were co-cultured with SAECs showed a reduction in migration capability at 24 and 48 hours. These data indicate that normal lun