SUPPLEMENTARY MATERIALS

Supporting methods

Cell migration assay

Cell migration was measured by Transwell assay (6.5 mm diameter; 8 mm pore size polycarbonate membrane; Corning Costar). After being treated as indicated for 48 h, A4549 cells were suspended in 0.5 ml serum-free medium and seeded in the upper compartment, whereas the lower compartment was loaded with 0.8 ml DMEM containing 10% FBS. After being cultured for an additional 24 h at 37°C, the cells that migrated into the lower chamber were fixed in 4% paraformaldehyde, stained with crystal violet and counted in five random fields under an inverted phases-contrast microscope (´40, Olympus).

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Fig. S1. Sorafenib represses TGF-b-dependent reporter activity in different cell types. HEK 293T, A549 or AML12 cells seeded in 24-well plates were transiently transfected with 200 ng of the (CAGA)12-Lux reporter. After 24 h of transfection, the medium was replaced with serum-free medium supplemented with TGF-b1 (5 ng/ml) in the absence or presence of sorafenib (5 mM), and cells were incubated for an additional 12 h before harvesting. As an internal control, 5 ng of pRL-SV40 plasmid expressing Renilla luciferase was included in each well.

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Fig. S2. Sorafenib inhibits the migration of A549 epithelial cells. A549 cells were treated with TGF-b1 (10 ng/ml) and sorafenib (5 mM) for 48 h, after which time cell migration was evaluated using a transwell assay. The asterisks denote significant differences (p<0.01) within experiments, as determined by the Student’s t-test.

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Fig. S3. Sorafenib inhibits the expression of TGF-b1-induced pro-fibrotic genes and reduces ECM production in alveolar epithelial cells. Following treatment with TGF-b1 (10 ng/ml) in the absence or presence of sorafenib (5 mM) for 48 h, real-time qPCR was performed to determine the effect of sorafenib on the expression levels of (A) the collagen family (alpha 1 chains of collagen type I, III and IV), (B) the pro-fibrotic gene CCN-2 and (C) the matrix metalloproteinases (MMP-2 and MMP-9). *, p<0.05; **, p<0.01, as determined by the Student’s t-test.

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Table S1 Primers used for real-time qPCR (h, m and r indicate human, mouse and rat species, respectively).

Target Gene / Primer Sequences (forward, reverse) / References
Bad (m) / F: 5’-AGAGTATGTTCCAGATCCCAG-3’;
R: 5’-GTCCTCGAAAAGGGCTAAGC-3’ / Yao et al., 2010
Bax (m) / F: 5’-GCTCTGAACAGATCATGAAG-3’;
R: 5’-GATGGTCACTGTCTGCCATG-3’ / Yao et al., 2010
Caspase-3 (m) / F: 5’-AGTCAGTGGACTCTGGGATC-3’;
R: 5’-GTACAGTTCTTTCGTGAGCA-3’ / Yao et al., 2010
CCN-2 (m) / F: 5’-CTGGGCAAACGTGTCTTC-3’
R: 5’-TGTGTGACGAGCCCAAGGA-3’ / Haydont et al., 2008
CCN2 (h) / F: 5’-GAGGAAAACATTAAGAAGGGCAA-3’
R: 5’-CGGCACAGGTCTTGATGA-3’ / Namba et al., 2010
Claudin (m) / F: 5′-GACTCCTTGCTGAATCTGAAC-3
R: 5′-CACCTCCCAGAAGGCAGAG-3′ / Dooley et al., 2008
Collagen I a1 (m) / F: 5’-CAAGAAGACATCCCTGAAGTC-3’
R: 5’-ACAGTCCAGTTCTTCATTGC-3’ / Rabani et al., 2010
Collagen I a1 (r) / F: 5’-TGCTCCTCTTAGGGGCCA-3’
R: 5’-CGTCTCACCATTAGGGACCCT-3’ / Espira et al., 2009
Collagen III a1 (r) / F: 5’-GGTTTCTTCTCACCCTGCTTCA-3’
R: 5’-GGTTCTGGCTTCCAGACATC-3’ / Espira et al., 2009
Collagen IV a1 (m) / F: 5’-CAGATTCCGCAGTGCCCTA-3’;
R: 5’-GGAATAGCCGATCCACAGTGAG-3’ / Veron et al., 2010
Collagen IV a1 (r) / F: 5’-AGCGAGATGTTCAAGAAG-3’
R: 5’-TGGACAGTGAGGTACACA-3’ / Espira et al., 2009
E-cadherin (h) / F: 5’-TGCCCAGAAAATGAAAAAGG-3’
R: 5’-GTGTATGTGGCAATGCGTTC-3’ / Namba et al., 2010
E-cadherin (m) / F: 5'-GCACTCTTCTCCTGGTCCTG-3'
R: 5′-TATGAGGCTGTGGGTTCCTC-3’ / Oba et al., 2010
FSP-1 (m) / F: 5′-GAAGCTGCATTCCAGAAGGTGA-3′
R: 5′-CATCATGGCAATGCAGGACA-3′ / Copple et al., 2010
GAPDH (h) / F: 5’-TGACTTCAACAGCGACACCCA-3’
R: 5’-CACCCTGTTGCTGTAGCCAAA-3’ / Liu et al., 2012
GAPDH (m) / F: 5’-TGTGTCCGTCGTGGATCTGA-3’
R: 5’-CCTGCTTCACCACCTTCTTGA-3’ / Yao et al., 2010
GAPDH (r) / F: 5’-TGCACCACCAACTGCTTAGC-3’
R: 5’-GGCATGGACTGTGGTCATGAG-3’ / Espira et al., 2009
MMP-2 (h) / F: 5’-AGTCTGAAGAGCGTGAAG-3’
R: 5’-CCAGGTAGGAGTGAGAATG-3’ / Namba et al., 2010
MMP-2 (m, r) / F: 5’-CCGAGGACTATGACCGGGATAA-3’
R: 5’-CTTGTTGCCCAGGAAAGTGAAG-3’ / Popov et al., 2008
MMP-9 (h) / F: 5’-TGACAGCGACAAGAAGTG-3’
R: 5’-CAGTGAAGCGGTACATAGG-3’ / Namba et al., 2010
MMP-9 (m, r) / F: 5’-TGGAACTCACACAACGTCTTTCA-3’
R: 5’-TCACCCGGTTGTGGAAACTC-3’ / Tsuda et al., 2010
MMP-13 (m, r) / F: 5’-GGTTGAGCCTGAACTGTTTTTGA-3’
R: 5’-CTCGTATGCAGCATCCACATG-3’ / Wang et al., 2010
procollagen type I (m) / F: 5’-acctgtgtgttccctactca-3’;
R: 5’-gactgttgccttcgcctctg-3’ / Ueberham et al., 2003
procollagen type III (m) / F: 5’-aatggtggttttcagttcagc-3’;
R: 5’-tggggtttcagagagtttggc-3’ / Ueberham et al., 2003
Snail (h) / F: 5’-GCCTAGCGAGTGGTTCTTCT-3’
R: 5’-TAGGGCTGCTGGAAGGTAAA-3’ / Namba et al., 2010
Slug (h) / F: 5’-GAGCATTTGCAGACAGGTCA-3’
R: 5’-ACAGCAGCCAGATTCCTCAT-3’ / Namba et al., 2010
a-SMA (m) / F: 5′-CCACCGCAAATGCTTCTAAGT-3′
R: 5′-GGCAGGAATGATTTGGAAAGG-3′ / Copple et al., 2010
TGF-b1 (h) / F: 5’-AAGGACCTCGGCTGGAAGTG-3’
R: 5’-CCGGGTTATGCTGGTTGTA-3’ / Namba et al., 2010
TGF-b1 (m) / F: 5’-TCGACATGGAGCTGGTGAAA-3’
R: 5’-GGGACTGGCGAGCCTTAGTT-3’ / Klein et al., 2009
TIMP-1 (m) / F: 5’-CATCCTCTTGTTGCTATCAC-3’
R: 5’-TGAATTTAGCCCTTATGACCAG-3’ / Rabani et al., 2010

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Supplementary References

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Dooley S, Hamzavi J, Ciuclan L, Godoy P, Ilkavets I, Ehnert S, et al. Hepatocyte-specific Smad7 expression attenuates TGF-beta-mediated fibrogenesis and protects against liver damage. Gastroenterology 2008; 135: 642-659.

Espira L, Lamoureux L, Jones SC, Gerard RD, Dixon IM, Czubryt MP. The basic helix-loop-helix transcription factor scleraxis regulates fibroblast collagen synthesis. J Mol Cell Cardiol 2009; 47: 188-195.

Haydont V, Riser BL, Aigueperse J, Vozenin-Brotons MC. Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation: a role for CCN2 and low concentration of TGF-beta1. Am J Physiol Cell Physiol 2008; 294: C1332-1341.

Klein J, Gonzalez J, Duchene J, Esposito L, Pradère JP, Neau E, et al. Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy. FASEB J 2009; 23: 134-142.

Liu H, Zhu L, Liu B, Yang L, Meng X, Zhang W, Ma Y, Xiao H. Genome-wide microRNA profiles identify miR-378 as a serum biomarker for early detection of gastric cancer. Cancer Lett 2012; 316: 196-203.

Namba T, Tanaka KI, Ito Y, Hoshino T, Matoyama M, Yamakawa N, et al. Induction of EMT-like phenotypes by an active metabolite of leflunomide and its contribution to pulmonary fibrosis. Cell Death Differ 2010; 17: 1882-1895.

Oba S, Kumano S, Suzuki E, Nishimatsu H, Takahashi M, Takamori H, et al. miR-200b precursor can ameliorate renal tubulointerstitial fibrosis. PLoS One 2010; 5: e13614.

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Veron D, Reidy KJ, Bertuccio C, Teichman J, Villegas G, Jimenez J, et al. Overexpression of VEGF-A in podocytes of adult mice causes glomerular disease. Kidney Int 2010; 77: 989-999.

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