Gsk 3β

TSA injection (sulfotanshinone sodium injection, 5 mg/mL; First Biochemical Pharmaceutical Co. Ltd., Shanghai, China) was used in the in vivo experiments. TSA monomer (Sigma, St. Louis, MO), a lyophilized powder (99.99% purity), which was first dissolved in dimethyl sulfoxide, and then diluted with phosphate-buffered saline (PBS) to the required concentration, was used in the in vitro experiments. Antibodies used for immunoblotting and/or immunohistochemistry were as follows: mouse anti-human monoclonal β-catenin (Abcam, Cambridge, MA, USA), rabbit anti-human polyclonal glycogen synthase kinase-3 beta (GSK-3β; Epitomics, Burlingame, CA, USA), rabbit anti-human polyclonal SOX2 (Epitomics, Burlingame, CA, USA), mouse anti-human monoclonal OCT4 (Abcam, Cambridge, MA, USA), mouse anti-human monoclonal Nanog (Abcam, Cambridge, MA, USA), rabbit anti-human polyclonal VEGF (Abcam, Cambridge, MA, USA), mouse anti-human monoclonal CD34 (Abcam, Cambridge, MA, USA), and mouse anti-human monoclonal actin (Beyotime, Shanghai, China).

Whole-cell extracts were prepared by scraping in 0.2–0.3 mL of RIPA lysis buffer as described earlier (Tsai et al. 2015 (link)). Nuclear and cytoplasmic fractions were harvested by using the Nuclear Extract Kit and following the manufacturer’s instructions (Active Motif, Carlsbad, CA). Protein concentrations were determined by Bradford method (BioRad, Hercules, CA). Equal amounts of protein from each treatment were diluted with loading buffer, boiled, and separated on 10 or 15 % gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes. After a short blocking with non-fat milk, the membranes were probed with appropriate primary antibodies, including AHR, ARNT (Santa Cruz), vimentin, β-catenin, N-cadherin, E-cadherin, claudin-1 (Genetex), occludin (Proteintech), ZO-1 (Invitrogen), lamin A/C, GSK3β phospho-Ser-9 and phospho-Tyr-216, GSK3β (Epitomics), and β-actin (Sigma), followed by an incubation with horseradish peroxidase-conjugated secondary antibodies. Immunocomplexes were visualized using the enhanced chemiluminescence (ECL) detection reagent (Millipore). Band intensities were determined by densitometry analysis using Gel-Pro Analyzer software (Media Cybernetics, Warrendale, PA).

Cells were lysed in RIPA cell lysis buffer (Beyotime, Shanghai, China) in the presence of protease inhibitor cocktail (Biotool, Huston, USA) and PMSF (Beyotime). Protein concentration was quantified by BCA protein assay kit (Beyotime). 50μg of the extracts were separated on 10% SDS-PAGE and transferred to PVDF membrane. Membrane was then blocked with 5% non-fat milk and incubated overnight with the following primary antibodies, respectively, which are Keratin10 (Abcam, Cambridge, USA), Involucrin (Proteintech, Wuhan, China), OCT4 (Proteintech), SOX2 (Proteintech), GSK-3β (Abcam), β-catenin (Proteintech), non-phospho β-catenin (Cell Signaling, Boston, USA) and LEF1 (Proteintech) followed by incubation with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h. Hybridization signal was detected by enhanced chemi-luminescence (ECL) (ThermoFisher, MA,USA) according to the manufacturer's instructions. GAPDH (ZSGB-BIO, Guangdong, China) was used as reference protein and determined following the same procedure as above.