P gsk 3βtyr216

5-FU and Sal were purchased from Sigma Aldrich (St. Louis, MO, USA). A stock solution of 25 mg/ml 5-FU and 25 mM Sal which prepared using dimethyl sulfoxide (DMSO) were stored in the dark at −20°C. The final 5-FU and Sal concentrations used in the experiments were prepared from the stock solutions by dilution in DMEM-h. The antibodies CD133 (Miltenyi, Germany) and EPCAM (eBioscience, USA) were used for flow cytometric analysis, β-actin(Santa Cruz, CA, USA), E-cadherin (Abcam, USA), vimentin (Abcam, USA), p-GSK-3β-Tyr216(Santa Cruz, CA, USA), p-β-catenin (Cell Signaling Technology, USA) and active β-catenin (Cell Signaling Technology, USA) for Western blotting, active β-catenin (Cell Signaling Technology, USA) for immunofluorescence, CD133 (Bioss, China), EPCAM (eBioscience, USA), E-cadherin (Abcam, USA), vimentin (Abcam, USA) and active β-catenin (Cell Signaling Technology, USA) for immunohistochemistry.

Western blotting analysis was carried out according to a previously published protocol (Lehmann et al., 2012 (link); Tschuor et al., 2016 (link)). Primary antibodies used in the present study included GSK-3β (Cell Signaling Technology, Inc.), p-GSK-3β^Ser9 (Cell Signaling Technology, Inc.), p-GSK-3β^Tyr216 (Santa Cruz Biotechnology, Inc.), PCK1 (Cell Signaling Technology, Inc.), PCK2 (Cell Signaling Technology, Inc.), AKT (Cell Signaling Technology, Inc.), p-AKT (Cell Signaling Technology, Inc.), non-phospho (active) β-catenin (Cell Signaling Technology, Inc.), c-Myc (Cell Signaling Technology, Inc.), cyclinD1 (Cell Signaling Technology, Inc.), PCNA (Cell Signaling Technology, Inc.) and MMP7 (Cell Signaling Technology, Inc.). GAPDH (Cell Signaling Technology, Inc.) was used as a loading control. The immunoreative bands were visualized with enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific, Waltham, MA, United States). Densitometric analysis of signal intensity was performed using Image J software (NIH, Bethesda, MD, United States).

Acrylamide, AO, bovine serum albumin (BSA), bromophenol blue, CQ, 4,6-diamidino-2-phenylindol (DAPI), DMBA, ethidium bromide, JC-1 iodide, 3-methyladenine (3-MA), 2-mercaptoethanol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sodium dodecyl sulphate (SDS), N,N,N’,N’-tetramethylene diamine (TEMED) and Trizol were acquired from Sigma Chemical Company, St. Louis, MO, USA. Power SYBR^® Green PCR master mix was obtained from Applied Biosystems, California, USA. Antibodies for Akt, β-actin, β-catenin, cleaved caspase-3, cleaved caspase-9, cytochrome c, GSK-3β, p-GSK-3β^Ser9, p-GSK-3β^Tyr216, PI3K, and Gapdh were purchased from Santa Cruz Biotechnology, USA. Antibodies for ATG5, Bax, Bcl-2, Beclin-1, Histone H2B, LC-3, p-Akt^Ser473, p-β-catenin^{Ser33,Ser37,Thr41}, and p-β-catenin^Ser552 as well as ELISA kits were from Cell Signaling Technology, USA. Alexafluor-488 conjugated anti-rabbit antibody was obtained from Molecular Probes, Inc. (Eugene, OR, USA). Annexin V-FITC, propidium iodide (PI) kit and p62 antibody were purchased from BD Biosciences (San Diego, CA). Nimbolide was obtained from M/s Asthagiri Herbal Research Foundation, Chennai, India. FuGENE transfection reagent was procured from Promega. Oligonucleotide primers were purchased from Sigma Genosys, San Ramon, USA. All other reagents used were of analytical grade.