Amphiregulin

Monolayer cultures of respective cell lines at an 80–90% confluence were lysed using 100 μl of RIPA buffer (Thomas Scientific). Tris-glycine (Bio-Rad) gels were loaded with 50–100 μg of lysates. After running gel electrophoresis, the gel was transferred to a nitrocellulose membrane for 2 hours. The membrane was blocked for 1 hour in 5% BSA or 5% skim milk at 4°C. The membrane was then washed 3 times with 1xTTBS and incubated overnight with the primary antibody at 4°C. Primary antibodies of coagulation factor III, IGFBP-3, DPP IV, PDGF-A, endothelin I, CXCL16, TIMP-1, amphiregulin, endostatin, angiogenin and GM-CSF were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies for STAT3, pSTAT3, E-cadherin, Vimentin, Oct-4, VEGF and β-actin were purchased from Cell Signaling Technology (Danvers, MA). After incubation with the secondary antibodies conjugated with horseradish peroxidase (HRP), the protein bands were developed with the chemiluminescent reagents. The blot images were taken by using the Bio-rad molecular imager gel doc XR system. Western blot protein band density was measured by Image J software and presented after normalized by the house keeping protein β-actin. Normal FHC (CRL-1831) colon cell line was used as a reference for the protein expression comparison by measuring band intensity as a 1.0.

The plasmid of Myc/His-tagged Sirt1 was constructed as described previously [23 (link)]. SIRT1-targeting lentivirus (SH3001-07) and non-targeting lentivirus were purchased from ATCGbio Life technology Inc. (Vancouver, Canada). The nucleotide sequences of siRNAs used are summarized in Supplementary Table 1. MMP-3 inhibitor (sc-311431) and antibodies against SIRT1, Myc tag, Amphiregulin, MMP3, SDF1, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A recombinant peptide of human MMP-3 (513-MP-010) was purchased from R&D Systems (Minneapolis, MN).