P mtor

EJ cells, HEK 293 cells, pcDNA3.1(−)-myc-RI, pGEX-4 T-RI and pEGFP-C1-RI plasmids were conserved and prepared by our laboratory. pGEX-4 T-1 was bought from GE Healthcare China. pEYFP-N1vector was from Clontech. pCMV-3xflag-CMVTM-10 was purchased from Sigma. BALB/C nude (nu/nu) mice were obtained from Beijing HFK Bioscience Company (Beijing, PR China). FBS was from TBD Science (Tianjin, PR, China). DMEM/High glucose medium, RPMI 1640 medium and G418 were purchased from Gibco-BRL (Carlsbad, CA, USA). Lipofectamine 2000 was bought from Invitrogen, Inc., (Carlsbad, California). Monoclonal mouse antibody of anti-human ILK was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-human β-actin, CD31 antibody, Monoclonal primary rabbit antibody of anti-human PI3K, p-PI3K, PTEN, p- PTEN, Akt, p-Akt, GSK3β, p-GSK3β, mTOR, p-mTOR and β-catenin were obtained from Bioworld Technology, Inc. (St. Louis, USA). The rest of the primary antibodies are from Proteintech Group, Inc (Chicago, IL, USA). Cell Counting Kit-8 was bought from Genview Scientific, Inc (Craigieburn, VIC, AUS).

Curcumin, Y15, 15d-PGJ2 and PD68235 were obtained from Sigma-Aldrich (St Louis, MO, USA). U0126 was obtained from Cell Signaling Technology (Danvers, MA, USA). Imatinib and fasudil were obtained from Nanjing EnoGene Biotechnology (Nanjing, China). Rapamycin was obtained from Xi'an Helin Biological Engineering (Xi'an, China). All these compounds were dissolved in dimethylsulfoxide (DMSO; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for experiments. Recombinant rat PDGF was obtained from Cell Sciences (Canton, MA, USA). Primary antibodies against VEGF, p-PI3K, PI3K, p-AKT and AKT were obtained from Nanjing EnoGene Biotechnology (Nanjing, China). Primary antibodies against α-smooth muscle actin (α-SMA), α(I) procollagen, fibronectin, p-PDGF-βR, PDGF-βR, p-FAK, FAK, GTP-RhoA and total-RhoA were obtained from Epitomics (San Francisco, CA, USA). Primary antibodies against PPAR-γ, p-ERK and ERK were obtained from Cell Signaling Technology. Primary antibodies against HIF-1α, VEGF-R2, p-mTOR, mTOR, p-p70S6K, p70S6K and β-actin were obtained from Bioworld Technology (Nanjing, China).

The protein extracted from HCC Cells or tissues were lysed in ice-cold radioimmunoprecipitation assay buffer and then incubated with primary antibodies overnight at 4°C. The primary antibodies are as follows: Tollip (1:1,000, ab37155, Abcam), Total-AKT (1:1,000, 4691, Cell Signaling Technology), p-AKT (1:1,000, 4046, Cell Signaling Technology), Total-mTOR (1:1,000, BS1555, Bioworld Technology), p-mTOR (1:1,000, BS470, Bioworld Technology), E-Cadherin (1:1,000, ab231303, Abcam), N-Cadherin (1:1,000, ab76011, Abcam), Snail (1:1,000, ab180714, Abcam), Vimentin (1:1,000, ab92547, Abcam), and GAPDH (1:1,000, SC-25778, Santa Cruz). The relative expression was obtained using a FluorChem E Imager (ProteinSimple, FluorChem E), and membranes were treated with enhanced chemiluminescence reagents (170-5061; Bio-Rad). The results were shown after normalizing against GAPDH gene expression.