Gel image system

The RNA extraction, cDNA synthesis and PCR reaction were performed as previously described [64 (link)]. These PCR products were analyzed by 1.5% agarose gel electrophoresis. The density of each band was quantified after normalization to the actin control band using the Gel Image System (Scion Corporation, MD). All the experiments were performed duplicate for three times. The error bars shown in the relevant figures indicated the standard deviation of the quantification results in all experiments. The primers used in this study for GDF15, CD44, and actin are listed in Supplemental Table S1.

Confluent cells were collected and lysed in RIPA buffer (Genestar Biotechnology, Taiwan). In total, 30 μg of proteins was separated by 10% sodium dodecylsulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Life Science, Carlsbad, CA, USA). After blocking, the membranes were hybridized with specific primary antibodies followed by incubation with secondary antibodies conjugated to horseradish peroxidase (HRP). The protein images were detected using the Western Blotting Plus Chemiluminescence Reagent (Thermo Scientific, Waltham, MA, USA). The density of each protein band was determined after normalization to an actin control band using the Gel Image System and Image J software (Scion Corporation, Torrance, MD, USA). The primary antibodies used included anti-ANXA2 (R&D System, Minneapolis, MN USA), anti-Akt, anti- Akt S473p, anti-Akt T308p, anti-Snail, anti-TWIST, anti-E-cadherin, and anti-N-cadherin (all from Genetex, Irvine, CA, USA). Protein images were detected using Western Blotting Plus Chemiluminescence Reagent (Thermo Scientific, Waltham, MA, USA). The density of each protein band was determined after normalization to an actin control band using the Gel Image System and Image J software.

The protein extraction and western blot analysis were performed as previously described [65 (link)]. Briefly, cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was hybridized with primary antibodies and then incubated with horseradish peroxidase-conjugated secondary antibodies. The membranes were developed using an ECL developing solution (Amersham Pharmacia Biotech, Piscataway, NJ, USA) followed by autoradiography. All the experiments were performed three times independently and that typical results were shown. Using the Gel Image System (Scion Corporation, MD), the density of each band was quantified after normalization to the GAPDH control band. The error bars shown in the relevant figures indicated the standard deviation of the quantification results in all experiments. The primary antibodies used in this study, including antibodies against GDF15, CD44, ALDH1, Nestin, ph-SMAD1/5, SMAD1/5, ph-SMAD3, SMAD3, and GAPDH, are listed in Supplemental Table S2.