1. Immunosuppression-induced bronchial epithelial–mesenchymal transition: A potential contributor to obliterative bronchiolitis 
Valerie M. Felton, Landon J. Inge, PhD, Brigham C. Willis, MD, Ross M. Bremner, MD, Michael A. Smith, MD 
The Journal of Thoracic and Cardiovascular Surgery 
Volume 141, Issue 2, Pages (February 2011)
DOI: /j.jtcvs
Copyright © 2011 The American Association for Thoracic Surgery Terms and Conditions

2. Figure 1
Morphology of immunosuppressive drug treated bronchial epithelium. RL-65 cells normally exhibit consistent round shape and tightly junctioned cobblestone monolayer. In presence of transforming growth factor β1 (TGF), however, cells began to flatten, elongate, and lose cell–cell adhesions, adopting more fibroblastlike morphology. Similar changes were seen in response to cyclosporine (CSA), mycophenolic acid (MMF), and sirolimus (RAPA). Azathioprine (AZA) elicited elongated and inconsistent cell shape with noticeably less loss of cell–cell adhesions. Cells treated with tacrolimus (TAC) and prednisone (PRED) were indistinguishable from those treated with vehicle (VEH).
The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs )
Copyright © 2011 The American Association for Thoracic Surgery Terms and Conditions

3. Figure 2
Immunoblot analysis of epithelial and mesenchymal markers. Cell lysates were analyzed for epithelial markers aquaporin 5 (AQP5) and E-cadherin. Aquaporin 5 expression ceased in presence of transforming growth factor β1 (TGF) and in cells treated with cyclosporine (CSA). Cells treated with tacrolimus (TAC), azathioprine (AZA), mycophenolic acid (MMF), and sirolimus (RAPA) all demonstrated significant loss of aquaporin 5 expression. Expression of E-cadherin remained consistent with control cells for transforming growth factor β1 and all drug treatments. Expression of mesenchymal marker and ECM protein fibronectin was greatly increased in response to transforming growth factor β1. Cyclosporine and mycophenolic acid elicited increased expression levels of fibronectin, similar to that of transforming growth factor β1. Azathioprine-treated cells showed slight increase in both fibronectin and vimentin. Prednisone-treated cells exhibited similar expression profile to that of vehicle-treated cells.
The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs )
Copyright © 2011 The American Association for Thoracic Surgery Terms and Conditions

4. Figure 3
Immunofluorescence localization of epithelial and mesenchymal markers. Actin cytoskeleton of vehicle-treated RL-65 cells showed intact cortical actin ring throughout cell monolayer. Treatment with transforming growth factor β1 (TGF) resulted in loss of actin ring and formation of visible stress fibers. Cyclosporine (CSA), mycophenolic acid (MMF), and sirolimus (RAPA) treatments mimicked cytoskeletal rearrangements of transforming growth factor β1. Azathioprine (AZA)–treated cells maintained their cortical actin ring; however, overall shape of the ring seemed to be elongated and irregular compared with VEH-treated cells. Transforming growth factor β1, cyclosporine, mycophenolic acid, and sirolimus also resulted in loss of E-cadherin (ECAD) localization at cell membrane relative to vehicle-treated cells, whereas cells exposed to tacrolimus (TAC) and prednisone (PRED) maintained cortical actin ring and membrane-associated E-cadherin expression. Transforming growth factor β1 treatment greatly increased fibronectin deposition, as did cyclosporine, mycophenolic acid, and sirolimus treatments. AZA-treated cells expressed increased fibronectin compared with VEH while also maintaining ECAD expression. Tacrolimus- and prednisone-dosed cells maintained baseline levels of fibronectin expression.
The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs )
Copyright © 2011 The American Association for Thoracic Surgery Terms and Conditions

5. Figure 4
Collagen production. Medium harvested from cell cultures was analyzed for presence of soluble collagen and normalized to total protein from same cultures. Cells treated with vehicle (VEH) exhibited virtually no collagen secretion, which we scaled to 1. Cells treated with transforming growth factor β1 (TGF) had nearly 5-fold increase in collagen production relative to vehicle-treated cells. Cells treated with cyclosporine (CSA) and sirolimus (RAPA) showed largest increases in collagen production, both at similar levels to transforming growth factor β1–treated cells. Cells treated with mycophenolic acid (MMF) and azathioprine (AZA) exhibited 3- and 4-fold increases in collagen production, respectively. Cells treated with tacrolimus (TAC) and prednisone (PRED) were comparable to vehicle-treated cells. Asterisk indicates significant difference from vehicle, P < .05.
The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs )
Copyright © 2011 The American Association for Thoracic Surgery Terms and Conditions

6. Figure 5
Transforming growth factor β1 (TGF) production by bronchial epithelial cells and anti–transforming growth factor β1 rescue. A, Presence of transforming growth factor β1 in culture medium samples was assayed by enzyme-linked immunosorbent assay. Transforming growth factor β1–positive control showed 1.5-fold increase in present transforming growth factor β1. Treatments with cyclosporine (CSA) and sirolimus (RAPA) elicited approximately 1.0-fold increases in transforming growth factor β1 production relative to cells treated with vehicle (VEH), whereas azathioprine (AZA) and mycophenolic acid (MMF) treatments resulted in approximately 0.5-fold increase. Cells treated with tacrolimus (TAC) and prednisone (PRED) were comparable to vehicle-treated cells. B, To demonstrate immunosuppressive-induced transforming growth factor β1 production as potential mechanism for immunosuppressive agent–induced epithelial–mesenchymal transition, we coincubated each immunosuppressive treatment with anti–transforming growth factor β1. As negative control, cells were treated with 2.5-ng/mL transforming growth factor β1 and 5-ng/mL anti–transforming growth factor β1. Transforming growth factor β1 alone served as positive control. Each immunosuppressive drug treatment coincubated with anti–transforming growth factor β1 resulted in maintenance of baseline fibronectin levels. Asterisk indicates significant difference from vehicle, P < .05.
The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs )
Copyright © 2011 The American Association for Thoracic Surgery Terms and Conditions