Mechanical stress induces lung fibrosis by epithelial-mesenchymal transition (EMT)
Nuria E. Cabrera-Benítez, PhD1-3*, Matteo Parotto MD1,4-6*, Martin Post PhD7,
Bing Han PhD1,4-6, Peter M. Spieth MD; PhD1,4-6, Wei-Erh Cheng MD1,4-6, Francisco Valladares
MD2,8, Jesús Villar MD; PhD1-,3, Mingayo Liu MD9, Masaaki Sato MD; PhD9, Haibo Zhang
MD; PhD1,4-6,10, Arthur S. Slutsky MD1,6,11,12
1
Keenan Research Center at the Li Ka Shing Knowledge Institute of St. Michael´s Hospital, Toronto,
Canada; 2CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Spain;3Multidisciplinary
Organ Dysfunction Evaluation Research Network, Hospital Universitario Dr. Negrin, Las Palmas de
Gran Canaria, Spain; 4Department of Anesthesia, 5Department of Physiology, 6Interdepartmental Division
of Critical Care Medicine, University of Toronto, Toronto, Canada; 7Lung Biology Program, Hospital
for Sick Children, Toronto, ON, Canada; 8Department of Anatomy, Pathology & Histology,
University of La Laguna, Tenerife, Canary Islands, Spain; 9Division of Thoracic Surgery, University
of Toronto, Toronto, Canada; 10State Key Laboratory of Respiratory Diseases, Guangzhou Institute
of Respiratory Diseases, First Hospital of Guangzhou Medical College, Guangzhou, China;
11
Adjunct Professor, King Saud University, Riyadh, Kingdom of Saudi Arabia; 12Department of
Medicine, University of Toronto.
Supplementary materials
Address for correspondence: Haibo Zhang, MD; PhD
Room 619, LKSKI
209 Victoria Street
Toronto, Ontario M5B1T8
Canada
Tel: (416) 864-6060 Ext. 77654
Fax: (416) 864-5277
Email: zhangh@smh.ca
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Materials and methods
Hydroxyproline measurement
Lung fibrosis was assessed by lung hydroxyproline content (1,2). Briefly, the right lung was
homogenized in 2 mL PBS, and a 1 mL aliquot was desiccated and hydrolyzed in 6 N HCl at
110ºC for 12 h. Every 25 µL aliquot was added to 1 mL buffer at pH 6.0 containing 1.4%
chloramine T (Sigma-Aldrich, Oakville, ON, Canada), 10% n-propanolol, and 0.5 M sodium
acetate, pH 6.0 and incubated for 20 min at room temperature. Erlich´s solution (1 M pdimethylaminobenzaldehyde [Sigma-Aldrich] in 70% n-propanolol, 20% perchloric acid) was
then added and incubated for 15 min at 65ºC. Hydroxylproline standard curve was prepared
using serial dilutions from 1 mg/mL stock solution (Sigma-Aldrich). Absorbance was read at 550
nm.
Reverse transcriptase-polymerase chain reaction (RT-PCR) assay
The mouse lung tissue was homogenized and human BEAS-2B cells were lyzed and total
RNA was extracted by using the Isol-RNA Lysis Reagent (5 PRIME, Markham, ON, Canada).
First-strand cDNA was prepared by using the SuperScript first-strand synthesis system
(Invitrogen), and RT-PCR was performed in a GeneAmp PCR system 2700 (Applied
Biosystems, Foster City, CA). The primers used to amplify the conserved regions of the genes of
interest in BEAS-2B cells and mouse lung tissue are shown in Tables 1 and 2, respectively. PCR
was performed by denaturation at 94ºC for 3 min or 95ºC for 10 min, followed by 35 - 50 cycles
of amplification (60ºC for 30 seconds – 2 min, 72ºC for 10 seconds – 2 min) and final extension
at 72ºC for 10 min. PCR products were resolved on a 1% agarose gel, stained with ethidium
bromide, photographed under ultraviolet illumination. The band intensity was quantified by
Image J 1.42q software (Scion Corporation, Frederick, MD).
References
1. Kim, K.K., Wei, Y., Szekeres, C., Kugler, M.C., Wolters, P.J., Hill, M.L., Frank, J.A.,
Brumwell, A.N., Wheeler, S.E., Kreidberg, J.A., et al. 2009. Epithelial cell alpha3beta1
integrin links beta-catenin and Smad signaling to promote myofibroblast formation and
pulmonary fibrosis. J Clin Invest 119:213-224.
2. Li P, Xiao HD, Xu J, Ong FS, Kwon M, Roman J, Gal A, Bernstein KE, Fuchs S.
Angiotensin-converting enzyme N-terminal inactivation alleviates bleomycin-induced lung
injury. Am J Pathol 2010;177:1113-1121
2
Supplemental Table 1. Human primers used and RT-PCR conditions
Primer
β-Catenin
TGF-β
Vimentin
α-SMA
Pro-SP-B
Cytokeratin-8
E-Cadherin
β-Actin
Forward
Primer (5´→3´)
Reverse
Primer (5 ´→3´)
TGGACCACAAGCAGAGTGCTGA
CCCTGTCACCAGCCCAAGG
AGGAGGTCACCCGCGTGCTA
CAGCGGGGTGGCCATGAGAA
GAGGAGATGCGGGAGCTGCG
ACGTACGTCACGCAGGGCAG
GCGGGGTCAGCACTTCGCAT
GCTCTGTGCTTCGTCACCCACG
TGGGGACATGTGGGAGCCGA
GAGCCCTGCAGAGCCAGCAA
AGCCGGCTCCAGGCTGAGAT
TGCATAGCCGCTGGTGGTCT
TGCACCGGTCGACAAAGGACA
GGGGTGTGTCAGCTCCTTGGC
GTCCGGCATGTGCAA
AGGATCTTCATGAGGTAGT
Annealing
Temperature
(ºC)
Cycles
(n)
55
35
55
35
55
35
55
35
37
50
55
35
55
35
55
35
TGF-β: Transforming growth factor-β; α-SMA: α-Smooth muscle actin; pro-SP-B: proSurfactant protein-B.
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Supplemental Table 2. Mouse primers used and RT-PCR conditions
Primer
β-Catenin
TGF-β
Vimentin
α-SMA
SP-B
Cytokeratin-8
E-Cadherin
β-Actin
Forward
Primer (5 ´→3´)
Reverse
Primer (5 ´→3´)
TCTCACCACCGCGAGGGCTT
AAGGCTTGGGGTCCACCACTG
GTGCCCGAACCCCCATTGCT
GTGCAGGTGCTGGGCCCTTT
ACCTCACTGCTGCCCTGCGT
CTCATCCTGCAGGCGGCCAA
AGCGTGGCTATTCCTTCGTGACT
CATGGTGCCTGGGTGCGAGGG
AGGACTTCTCTGAGCAACAG
ATGGCATCCTCAGGAAGA
ATGCAGGGCCTGGTGGAGGA
GCCTCAGCTCGGCTGCGATT
CCAGTTTCCTCGTCCGCGCC
GCTCCTTGGCCGGTGATGCT
TGTTACCAACTGGGACGACA
TCTCAGCTGTGGTGGTGAAG
Annealing
Temperature
(ºC)
Cycles
(n)
55
35
55
35
55
35
55
35
60
35
55
35
60
35
55
35
TGF-β: Transforming growth factor-β; α-SMA: α-Smooth muscle actin; SP-B: Surfactant
protein-B.
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