Western blot analysis

Western blotting was carried out as described previously [33, 54]. Briefly, cells or tissue homogenates were lysed in NP40 cell lysis buffer (Biosource, Camarillo, CA, USA) to extract total cellular proteins once the treatment was finished. Protein was measured by using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of proteins (50 μg/lane) were resolved by using 4-20% gradient SDS-PAGE (Life Technology). After transferring, blots of nitrocellulose membrane were blocked in 5% fat-free milk in 0.05% Tween-20, 20 mM phosphate-buffered saline, pH 7.4 (PBST), and then incubated with each one of the primary antibodies (1:500 or 1:2000 dilution), at 4°C overnight. After PBS washing, these blots were incubated with corresponding horseradish peroxidase–conjugated secondary antibodies (1:5000 dilutions) and developed using SuperSignal West Femto substrate (Thermo Fisher Scientific). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control for cellular protein. Relative protein levels present were calculated from the OD values, normalized against those for GAPDH. Antibodies against human p53 phosphorylated at Ser15, and against c-Myc, Klf4, Zeb1, and Oct4, were purchased from Cell Signaling Technology (Danvers, MA). Antibody for glucosylceramide synthase (GCS) was purchased from GeneScript (Piscataway, NJ). Antibodies for Puma, p21Waf1/Cip1, Bax, p53, E-cadherin, vimentin, TGF-β, β-catenin, and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX).