Gsk3α β

We employed antibodies against the following: IRβ (catalog 3025, Cell Signaling Technology), Laminin γ1 (catalog sc-5584, Santa Cruz Biotechnology), fibronectin (catalog ab2413, Abcam), type I collagen α2 (catalog 14695-1-AP, Proteintech), phospho-Ser9-GSK3β (catalog 9323, Cell Signaling Technology), GSK3α/β (catalog sc-56913, Santa Cruz Biotechnology), phospho-Ser539-eIF2Bε (catalog 44-530G, Thermo Fisher Scientific), eIF2Bε (catalog 3595, Cell Signaling Technology), phospho-Ser2448-mTOR (catalog 2971, Cell Signaling Technology), mTOR (catalog 2972, Cell Signaling Technology), phospho-Thr389-p70S6 kinase (catalog 9205, Cell Signaling Technology), p70S6 kinase (catalog 9202, Cell Signaling Technology), CBS (catalog sc-67154, Santa Cruz Biotechnology), CSE (catalog sc-135203, Santa Cruz Biotechnology), IGF-1 receptor (catalog 3027, Cell Signaling Technology), phospho-Thr202/Tyr204-Erk (catalog 4377, Cell Signaling Technology), Erk (catalog 9102, Cell Signaling Technology), phospho-Ser473-Akt (catalog 9271, Cell Signaling Technology), Akt (catalog 9272, Cell Signaling Technology), nephrin (catalog ab58968, Abcam), and actin (catalog A2066, MilliporeSigma). Commercial ELISA kits were used for measuring phospho-Tyr-1150/1151-IR (catalog 7082, Cell Signaling Technology) and phospho-Ser79-ACC (catalog 7986, Cell Signaling Technology).

The human NPC cell line CNE-1 was cultured and conserved by Sun Yat-sen University cancer center from 1982 and have been used in previous study [41 (link)], and the S18 cell line is a single cell clone of CNE-2 (a kind gift from Dr. Chao-Nan Qian in 2011, China) [55 (link)] and has also been used in previous study [22 (link)]. Both cell lines were authenticated by Applied Biosystems on Nov 16, 2012 via STR analysis. 293T cell line was obtained from the American Type Culture Collection in 2010. The cells were cultivated in DMEM medium supplemented with 10% fetal bovine serum in a 5% CO2 humidified atmosphere at 37°C. GAPDH, AKT, phospho-AKT (Ser473), phospho-AKT (Thr308), GSK3α/β, cleaved PARP, p27, Sox2, α-tubulin primary antibodies and a horseradish peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (California, CA, USA). Anti-phospho-FKHRL1, phospho-GSK3β, and chemiluminescence reagents were obtained from Cell Signaling Technology (Beverly, MA, USA). Hoechst 33342, Fumitremorgin C (FTC), DAPI, MTT and DMSO were acquired from Sigma-Aldrich (St. Louis, MO, USA).

The following antibodies were used: NF-κB p65 (western blot 1:1,000, rabbit, Cell Signaling, #8284), GAPDH (Western blot 1:3,000, mouse, Santa Cruz, #sc-47724), phospho (Ser 473) Akt (Western blot 1:1,000, rabbit, Cell Signaling, #9271), Akt (Western blot 1:1,000, rabbit, Cell Signaling, #9272), phospho (Ser21/9) GSK-3α/β (Western blot 1:1,000, rabbit, Cell Signaling, #9331), GSK-3α/β (Western blot 1:1,000, mouse, Santa Cruz, #sc-7291), phospho (Ser675) β-catenin (Western blot 1:1,000, rabbit, Cell Signaling, #4176), β-catenin (Western blot 1:1,000, mouse, BD Biosciences, #610154), Histone H3 (Western blot 1:1,000, rabbit, #4499), non-phospho (active) β-catenin (Western blot 1:500, rabbit, Cell Signaling, #8814), IκB-α (Western blot 1:1,000, rabbit, Santa Cruz, #sc-203), acetyl NF-κB p65 (Lys310) (Western blot 1:500, rabbit, Cell Signaling, #3045), and normal mouse IgG (Santa Cruz, #sc-2025).
Other reagents include the following: DMSO (Sigma Aldrich, #472301), ICG-001 (Tocris, #4505), XAV-939 (Tocris, #3748), IQ-1 (Tocris, #4713), FBS (Thermo Scientific, #SV30180.03), and recombinant IL-1β (Sigma Aldrich, #SRP3083). All other chemicals were of analytical grade.

Cells were lysed in RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM phenylmethylsulfonylfluoride, protease inhibitor mixture (Sigma, St. Louis MO, USA), 1 mM sodium orthovanadate). Equivalent amounts of protein lysate were separated by SDS-PAGE, transferred to PVDF membrane and incubated with the followed primary antibodies: β-actin, E-cadherin, GFP, β-catenin, GSK3-αβ (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-GSK3β (Cell Signaling, Danvers, MA, USA), DPEP1(Sigma, St. Louis MO, USA). The bound antibodies were visualized with a suitable secondary antibody conjugated with horseradish peroxidase using enhanced chemiluminescence (Amersham Bioscience, Pittsburgh, PA, USA).

3T3-L1 MBX cells (3 × 10⁴ cells/well) were seeded in 60 mm cell culture dishes and differentiated for 8 days. The cells were lysed in a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 0.25% sodium deoxycholate, 1 mM orthovanadate, aprotinin (10 μg/ml), and 0.4 mM phenylmethylsulfonyl fluoride at 4°C for 30 min. Equal amounts of total cellular protein (50 μg) isolated from the harvested cells were separated by SDS-PAGE and transferred onto a PVDF membrane (Choi et al., 2022 (link)). Specific proteins were detected using antibodies against PPARγ, CCAAT/enhancer-binding protein (C/EBP) α, phosphorylated protein kinase B (PKB/AKT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), C/EBPβ, β-actin and phosphorylated glycogen synthase kinase three α/β (GSK3 α/β) (Santa Cruz Biotechnology, Santa Cruz, CA, United States ). Antibodies against fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) and glucose transporter type 4 (GLUT4) were purchased from Cell Signaling Technology (Danvers, MA, United States).