Phosphatase inhibitor

Total cellular proteins were extracted in lysis buffer (1% NP-40, 0.1% sodium dodecyl sulfate, pH 7.3, 50 mM Tris, and 150 mM NaCl) with protease inhibitors (Roche) and phosphatase inhibitors (KeyGEN Biotech) for 30 min on ice and centrifuged at 15,000 rpm at 4°C. Nuclear and Cytoplasmic Protein Extraction kIt (KeyGEN Biotech) was used to separate the nuclear and cytoplasmic protein according to the manufacturer’s protocol. Western blots were carried out as previously described [52 (link)]. The primary antibodies used included anti-TCF8/ZEB1, anti-β-catenin, anti-p-GSK3β (S9), anti-GSK3β, anti-AKT, anti-p-AKT, anti-PTEN, anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-TCF1, anti-LEF1, anti-DVL2, anti-p-JNK, anti-p-ERK, anti-p-p38 (MAPK), anti-NF-κB (p65) , p-IκBα (all 1:1,000; Cell Signaling Technology), anti-OTUB1 (1:1,000; Abcam, Cambridge, MA), anti-GAPDH (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-β-actin (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Histone H3 (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA).

Western blotting analyses were performed by using standard methods. In brief, cells were harvested and lysed in the RIPA buffer containing protease inhibitors (Sigma-Aldrich) and phosphatase inhibitors (Keygen, China). Proteins were separated by SDS–polyacrylamide gel electrophoresis gels, and blotted onto PVDF membrane (Millipore). The membrane was probed with the first antibody listed in Supplementary Table 6 and then with the peroxidase-conjugated secondary antibody. GAPDH was used as a protein loading control. Western blotting bands were visualized by eECL Western Blot Kit (CWBIO Technology) and captured with ChemiDocTM CRS+ Molecular Imager (Bio-Rad). All blots in figures were accompanied by the locations of molecular weight/size markers. Original blotting images for the key components of PTEN-dependent pathways and EMT were shown in Supplementary Fig. 9.

Western blotting analyses were performed with standard methods. Briefly, cell pallets were lysed in the radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors (Sigma-Aldrich) and phosphatase inhibitors (Keygen, China). Proteins were separated by 10% SDS-PAGE gels, and blotted onto PVDF (polyvinylidene difluoride) membrane (Millipore). The membrane was probed with the specific antibodies (Table S6), and then with peroxidase-conjugated secondary antibodies. Beta-actin was used as a protein loading control. The bands were visualized by eECL Western Blot Kit (CWBIO Technology). The images were captured with ChemiDocTM CRS+ Molecular Imager (Bio-Rad).

HCC cells were suspended in serum-free media on a six-well plate at a density of 2.5 × 10⁵/ml. A total of 2 ml of cell suspension was utilized, containing different concentrations of drug or inhibitor. After cell attachment, the conditioned media were collected. Tumor samples were minced on ice in prechilled lysis buffer containing phenylmethylsulfonyl fluoride, protease inhibitors, and phosphatase inhibitors (KeyGen BioTech, China). The homogenized tissues and cell lysates were then centrifuged at 14,000 rpm at 4°C for 15 min. The BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL) was employed to determine the total protein density and to ensure that equal amounts of proteins were separated on the 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Boston, MA). After blocking with 5% nonfat milk for 1 hr. The membrane was incubated with the designated primary antibody at 4°C overnight. Immunodetection was performed using the Western-Light chemiluminescent detection system (Applied Biosystems, Foster City, CA) after incubation with the secondary antibody for 1 hr.

Cell pellets were lysed in RIPA buffer containing protease (Sigma-Aldrich) and phosphatase inhibitors (Keygen, China), and the protein concentration was determined using the BCA assay (Beyotime, Beijing, China). Proteins were separated by a 10% SDS-PAGE gel, and blotted onto a polyvinylidene difluoride membrane (Milipore, Billerica, MA, USA). The membrane was probed with the first antibody listed in S1 Table and then with the peroxidase conjugated secondary antibody. GAPDH and β-actin were used as protein loading controls. Western blotting bands were visualized by the eECL Western Blot Kit (CWBIO Technology) and captured with a ChemiDoc CRSt Molecular Imager (Bio-Rad).

Western blotting analyses were performed with standard methods. Briefly, cell pallets were lysed in the radio-immunoprecipitation assay buffer containing protease inhibitors (Sigma-Aldrich) and phosphatase inhibitors (Keygen, Nanjing, China). Proteins were separated by 10% SDS-PAGE gels, and blotted onto polyvinylidenedifluoride membrane (Millipore, Billerica, MA, USA). The membrane was probed with the specific antibodies (Supplementary Table 2), and then with peroxidase-conjugated secondary antibodies. GAPDH was used as a protein loading control. The bands were visualized by eECL Western Blot Kit (CWBIO Technology, Beijing, China). The images were captured with ChemiDocTM CRS+ Molecular Imager (Bio-Rad, Hercules, CA, USA).

Total cellular protein was extracted and lysed with RIPA lysis buffer with protease inhibitors (KeyGEN, Nanjing, China) and phosphatase inhibitors (KeyGEN, Nanjing, China). Proteins were separated with 10% SDS-PAGE gels and transferred to PVDF membranes (Merck Millipore, MA, USA). Membranes were blocked with 5% non-fat milk for 1h and then incubated with primary antibodies of PPARγ, ANGPTL4, MMP-2, IL-1β, GAPDH, and tubulin β (Bioword, Beijing, China) overnight at 4 °C. After being washed five times for five minutes, the membranes were incubated with the corresponding secondary antibodies for 1h at room temperature. The protein bands were visualized with an Enhanced Chemiluminescence (ECL) detection kit (Amersham, NJ, USA). The protein bands were analyzed using Image Lab Software (Bio-Rad, CA, USA).