Ser9 phosphorylated gsk3β

The natural product bergenin, MG132, 5-Fu, regorafenib, and cycloheximide (CHX) were obtained from Selleck Chemicals (Houston, TX, USA). The transfection reagent Lipofectamine^TM 2000 was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Antibodies against cytochrome c (#11940, 1:1000), Bax (#14796, 1:1000), phosphorylated (p)-GSK3β-Ser9 (#12456, 1:1000), p-Mcl-1-Ser159 (#39224, 1:1000), p-Akt-Ser473 (#4060, 1:1000), Akt (#4691, 1:1000), cleaved-caspase 3 (#9664, 1:1000), β-actin (#3700, 1:10,000), and cleaved-poly (ADP-ribose) polymerase (PARP) (#5625, 1:1000) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-Ki67 (#ab15580, 1:300) was purchased from Abcam (Cambridge, UK).

Total protein was extracted using radioimmunoprecipitation assay buffer according to the manufacturer’s protocol, and the protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Biotec). Protein samples were fractionated using 8–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% non-fat milk in TBS-T and incubated overnight at 4 °C with the following antibodies: FZD7 (Abcam); E-cadherin, N-cadherin, vimentin, SNAI2 (snail family transcriptional repressor 2), CD44, CD133 (prominin 1), nestin, SOX2 (SRY-box 2), GSK3β (glycogen synthase kinase 3 beta), β-catenin, c-MYC (Proteintech), and phosphorylated p-GSK3β (Ser9) (Cell Signaling Technology); the endogenous control was GAPDH (Proteintech), and the nuclear control was histone H3 (Abcam). The immune complexes were detected using enhanced chemiluminescence.

Soluble proteins from treated cells were harvested and lysed in solution containing 50 mM HEPES (pH 7), 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mM Na₃VO₄, 10 μM pepstatin, 10 μg/mL aprotinin, 5 mM iodoacetic acid, and 2 μg/mL leupeptin. Equal amounts of protein were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The membranes were then probed with the following primary antibodies: CD133 (#64326S, Cell Signaling Technology, Danvers, MA, USA), SOX2 (#23064 S, Cell Signaling Technology), β-catenin (#8480S, Cell Signaling Technology), Ser675-phosphorylated β-catenin (#4176S, Cell Signaling Technology), Ser9-phosphorylated GSK3β (#9336S, Cell Signaling Technology), GSK3β (#5676S, Cell Signaling Technology), PKA (#5842S, Cell Signaling Technology), GAPDH (#Ab103, Vazyme, Nanjing, China), and β-actin (#Ab101-03, Vazyme). Immunoreactivity was visualized by probing with the HRP-conjugated secondary antibody (#7074, Cell Signaling Technology) and detected using the ECL kit (#sc-2048, Santa Cruz, Santa Cruz, CA, USA). Western blot band intensities were quantified through densitometric analysis using ImageJ and normalized the band intensities with the loading control band. Original uncropped WB are given in Supplementary Material.