Snail

Cells were lysed in RIPA lysis buffer (Beyotime, Nanjing, China) with the protease inhibitor phenylmethanesulfonyl fluoride (Beyotime). Protein concentrations were determined using a BCA Protein Assay kit (Beyotime). Equal amounts of protein were loaded into each lane of an SDS-PAGE gel. Proteins were then separated with electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). Each membrane was blocked with 5% skimmed milk for 2 h and then incubated with antibodies against SHARP1 (1:500), E-cadherin (1:1000), N-cadherin (1:1000), vimentin (1:1000), Snail (1:1000), NOTCH1 (1:2500; Epitomics, Burlingame, CA), jagged 1 (1:10000), HES1 (1:2000; Epitomics), or β-actin (1:5000; Epitomics) at 4°C overnight. Peroxidase-linked secondary antibodies against rabbit (1:5000; Epitomics) were used to detect bound primary antibodies. Probed proteins were detected by enhanced chemiluminescent reagents. The data have been normalized to β-actin expression by densitometry and statistical data from at least 3 experiments is graphed to provide additional validation of results.

Flaccidoxide-13-acetate was supplied by Dr. Jui-Hsin Su. Rabbit anti-human Akt, FAK, MMP-2, MMP-9, MMP-13, mTOR, PI3K, TIMP-1, TIMP-2, uPA and phosphorylated Akt, mTOR, and PI3K were purchased from Cell Signaling Technology (Danvers, MA, USA). E-cadherin, N-cadherin, lamin A2, and Snail antibodies were supplied by Epitomics (Burlingame, CA, USA). HRP-conjugated goat anti-rabbit immunoglobulin (IgG) was obtained from Millipore (Bellerica, MA, USA). Thiazolyl blue tetrazolium bromide, streptomycin, penicillin, and DMSO were purchased from Sigma (St. Louis, MO, USA). Goat anti-rabbit and horseradish peroxidase-conjugated immunoglobulin IgG and polyvinylidene difluoride (PVDF) membranes were obtained from Millipore (Bellerica, MA, USA). Fetal bovine serum and DMEM were purchased from Biowest (Nuaillé, France).

The stems of Piper betle were collected in Pingtung County, Taiwan in July 2008, which were cultivated by local farmer and bornyl cis-4-hydroxycinnamate identified by Chi-I Chang, National Pingtung University of Science and Technology. Rabbit anti-human MMP-2, MMP-9, uPA, TIMP-1, TIMP-2, FAK, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, JNK, p-JNK, Jun, p-Jun, p38, p-p38, ERK, and p-ERK were obtained from Cell Signaling Technology (Danvers, MA, USA). GRB2, Rac, PKC, Ras, RhoA, MEKK3, MEKK7, N-cadherin, E-cadherin, Snail, and Lamin A2 antibodies were obtained from Epitomics (Burlingame, CA, USA). Dimethyl sulfoxide (DMSO), protease inhibitor cocktail, and rabbit anti-human β-actin antibodies were purchased from Sigma (St. Louis, MO, USA). PVDF (polyvinylidene difluoride) membranes and goat anti-rabbit and horseradish peroxidase-conjugated IgG were purchased from Millipore (Bellerica, MA, USA). Chemiluminescent horseradish peroxidase (HRP) substrate was purchased from Pierce (Rockford, IL, USA).