Vimentin

Fluorescent dyes (Cy2, Cy3, and Cy5) and reagents for 2D-DIGE were purchased from GE Healthcare (Uppsala, Sweden). Lipofectamine^® RNAiMAX Transfection Reagent and OPTI-MEM were purchased from Invitrogen (Waltham, MA, USA). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from USB Corp. (Cleveland, OH, USA). Propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human LGALS1 protein (rhLGALS1) was purchased from BioLegend, Inc. (San Diego, CA, USA). LGALS1, cyclin A2, cdk2, phosphor-ERK (Thr202), MMP-9, MMP-3, ZEB2, SNAI1, TWIST, E-cadherin, N-cadherin, and vimentin primary antibodies were purchased from Genetex Inc. (Hsinchu, Taiwan). Phospho-p38 MAPK (Thr180/Tyr182) and p38 MAPK primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Cyclin D2, cdk4, cyclin E, and p27 primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). ERK1/2 primary antibody was purchased from Promega Corp. (Madison, WI, USA). Anti-rabbit and anti-mouse immunoglobulin (Ig)G secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). All the chemicals and reagents used in this study were of analytic grade.

Total cellular protein was extracted by cells lysis buffer RIPA, which was added to a protease inhibitor mixture. Next, 30 ug of protein was separated by SDS-PAGE, and the protein was transferred onto a polyvinylidene fluoride (PVDF) membrane by the wet transfer method, and the protein – antibody complex was detected by immunoassay^(19). Primary antibodies used were as follows: TET1 (GeneTex, 1:1000), E-cadherin (GeneTex, 1:1000), Vimentin (GeneTex, 1:1000), N-cadherin (GeneTex, 1:1000), Beclin1 (GeneTex, 1:1000), LC3 (Beyotime, AF5225, 1:500), P62 (Beyotime, AF5312,1:500), and Atg5 (Beyotime, 1:500); GAPDH (Beyotime, AF0006, 1:1000). The second antibodies included HRP-goat anti-rabbit (Beyotime, AF0208, 1:1000) or HRP-goat anti-mouse (Beyotime, AF0216, 1:1000). Finally, the protein – antibody complexes were detected with an enhanced chemiluminescence (ECL) reagent.

Total protein was extracted from the cells. Approximately 25 μg sample was processed by 12% polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were placed in a blocking solution and slowly shaken for 2 h at 37°C. After blocking, the membranes were mixed with primary antibodies to PUMA (ab33906, 1:1000, Abcam, Cambridge, UK), Bax (#33-6400, 1:500, Invitrogen Inc., Carlsbad, CA, USA), Bcl-2 (#MA5-11757, 1:1000, Invitrogen), E-cadherin (GTX100443, 1:500, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), Vimentin (GTX100619, 1:1000, GeneTex), Snai1 (#14-9859-82, 1:500, Invitrogen), N-cadherin (ab70611, 1:2000, Abcam), CDC73 (#PA5-17159, 1:1000, Invitrogen) and GAPDH (ab8245, 1:2000, Abcam) overnight at 4°C. The membranes were then incubated with the secondary antibody to IgG labelled with horseradish peroxidase (ab6721, 1:1000, Abcam) for 2 h at 37°C. Protein signals were visualized using enhanced chemiluminescence. The protein levels were analyzed using ImageJ software. The relative expression of the target protein was obtained by calculating the ratio of optical density (OD) value of the target protein to that of an internal reference.