Supporting Methods
Cell migration assay. Cell migration was detected using a Transwell assay
system (Corning Coster; Cambridge, MA). After being treated for 48 h, cells
were trypsined and seeded in the upper compartment at the rate of 5х104
cells/well in 200 L of DMEM/F12 containing 0.5% NCS. The lower
compartment was filled with 600 L of DMEM/F12 containing 10% FBS. After
being cultured for 10 h, cells were stained with the Hematoxylin and Eosin
Staining Kit (Beyotime Institute of Biotechnology, China). The number of cells
migrated to the lower surface of the filter membrane was counted in 6 random
fields under an inverted phases-contrast microscope (x100).
PI staining and apoptotic analysis. After being treated with TGF-1 and
other reagents, cells in 35-mm dishes were trypsinized and fixed with 70%
ethanol for more than 1 h. Cells were pelleted and washed with PBS plus 20
mM EDTA. After being incubated with RNase (1 mg/mL) at 37 oC for 1 h, cells
were stained with PI (30 mg/mL). The nuclei were visualized at
400× magnification, and cells with nuclear condensation or fragmentation were
supposed to be the apoptotic cells. The apoptosis proportion was determined
as the rate of the number of apoptotic cells to whole cells.
Reporter plasmids and cell transfection. The luciferase reporters ARE-luc,
(CAGA)12-luc, (SBE)4-luc and FAST-1 plasmid were generous gifts from Dr.
Peter ten Dijke1,2. Cells were transiently transfected, and luciferase activities
(firefly luciferase and Renilla luciferase) were determined using the
Dual-Luciferase Reporter Assay System (Promega). In all reporter assays, the
Renilla luciferase activity was measured to correct for transfection efficiency.
The experiments were performed at least three times.
Supporting Fig. S1. SNAP inhibits EMT and cell migration. Mouse primary
hepatocytes were treated with SNAP (1 mM) and TGF-1 (5 ng/mL) for 48 h,
EMT was examined by cell morphological changes (a). b. AML12 cells and
mouse primary hepatocytes were treated with SNAP (1 mM) and TGF-1 (5
ng/mL) for 48 h, and cell migration was detected by transwell assay.
Supporting Fig. S2. SNAP inhibits TGF-1-induced apoptosis. AML12
cells were treated with SNAP (1 mM) and TGF-1 (5 ng/mL) for 24 h. a.
PI-stained cells were examined with fluorescence microscope and the
representative results were presented. The arrow indicates an exemplary
apoptotic cell. b. Cells were counted and the apoptotic rate was defined as the
number of apoptotic cells out of the total cells.
Supporting Fig. S3. TGF- type I receptor inhibitor, SB431542, blocks
STAT3 phosphorylation, apoptosis and EMT in AML12 cells. AML12 cells
were treated with TGF-1 (5 ng/mL) and SB431542 (2 M) for 4 h, and cell
lysates were immunoblotted with anti-STAT3 (Tyr705) and anti-STAT3 (a). b.
AML12 cells were treated with TGF-1 (5 ng/mL) and SB431542 (2 M) for 48
h, and cell apoptosis was detected by FACS assay. AML12 cells were treated
with TGF-1 (5 ng/mL) and SB431542 (2 M) for 48 h, and EMT was
examined by cell morphological changes (c) or immunoblotting of ZO-1,
E-cadherin and fibronectin (d).
Supporting Fig. S4. NO regulates TGF-1-induced Smad transcriptional
activity in AML12 cells. AML12 cells transfected with ARE-luc, RL-TK-luc and
FAST-1 were treated with SNAP (1 mM), SB431542 (2 M) and TGF-1 (5
ng/mL) for 16 h, luciferase activities were detected (a). b. AML12 cells
transfected with (CAGA)12-luc and RL-TK-luc were treated as described above.
c. AML12 cells transfected with (SBE)4-luc and RL-TK-luc were treated as
described above. For each assay, RL-TK-luc activity was used to normalize
the transfection efficiency and the results were presented as relative luciferase
values.
Supporting References
1. Piek E, Van Dinther M, Parks WT, Sallee JM, Böttinger EP, Roberts AB, Ten
Dijke P. RLP, a novel Ras-like protein, is an immediate-early transforming
growth factor-beta (TGF-beta) target gene that negatively regulates
transcriptional activity induced by TGF-beta. Biochem J. 2004 Oct 1;383(Pt
1):187-99.
2. Itoh S, Thorikay M, Kowanetz M, Moustakas A, Itoh F, Heldin CH, ten Dijke P.
Elucidation of Smad requirement in transforming growth factor-beta type I
receptor-induced responses. J Biol Chem. 2003 Feb 7;278(6):3751-61.