Sorafenib tosylate

All small molecule inhibitors were obtained from Selleck Chemicals (Houston, TX, USA), these inhibitors include: Sorafenib tosylate (#S1040), PI3K inhibitors: LY294002 (#S1105), Buparlisib (#S2247), Pictilisib (#S1065), Alpelisib (#S2814) and Akt inhibitors MK2206 (#S1078), Ipatasertib (#S2808), Afuresertib (#S7521). All chemicals formulated by their suppliers’ recommendations.

Griffipavixanthone was isolated from twigs of Garcinia esculenta as previously described [27 (link)]. Phosphate buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), Propidium iodide (PI), Bouin's solution and 5-FU were purchased from Sigma-Aldrich (St. Louis, MO, USA). Matrigel was purchased from BD (San Jose, CA, USA). Sorafenib Tosylate was purchased from Selleck Chemicals (Houston, TX, USA).

PB2, pentobarbital sodium salt, cyclopamine hydrate and Smoothened agonist (SAG) were purchased from Sigma‐Aldrich (Merck KGaA). Sorafenib tosylate was purchased from Selleck Chemicals. CCl4 was purchased from China Sinopharm International Corporation. TGF‐β1 was obtained from PeproTech. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hydroxyproline detection kits were purchased from Nanjing Jiancheng Bioengineering Institute. Oligonucleotide primers were synthesized by Generay Biotech Co., Ltd. The PrimeScript RT Reagent kit and SYBR Premix Ex Taq were purchased from TaKaRa Biotechnology. The primary antibodies used in the study are listed in Table 1. Anti‐goat or antimouse secondary antibodies were obtained from Dako. Dulbecco's Modified Eagle Medium (DMEM) and foetal bovine serum (FBS) were from HyClone (GE Healthcare). The Annexin V‐FITC apoptosis detection kit was purchased from BD Biosciences. Growth factor‐reduced Matrigel was purchased from BD Biosciences. PB2, sorafenib and cyclopamine were dissolved in DMSO (<0.1% [v/v]) for in vitro treatment.

Resveratrol was purchased from Sigma-Aldrich (St. Louis, MO). Sorafenib tosylate was purchased from Selleck (Selleck Chemicals, Shanghai, China). For cell treatment, the compounds were dissolved in dimethyl sulfoxide (DMSO; Gibco, Carlsbad, CA, USA) and then diluted in media with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) before use.
All cultured cells were purchased from the Chinese Academy of Sciences Committee Type Culture Collection cell bank. HCC-LM3, Huh-7, HepG2, SMMC-7721, Bel-7402, and LO2 were maintained in Dulbecco's modified Eagle's Medium (DMEM) with high glucose (Hyclone) supplemented with 10% FBS. QSG-7701 was cultured in RPMI-1640 with 10% FBS at 37°C in a humidified atmosphere of 5% CO₂.

Pemetrexed was purchased from LC Laboratories (Woburn, MA). FTY720 was purchased from Cayman Chemical Inc., (Ann Arbor MI). Sorafenib tosylate and all ERBB receptor and kinase inhibitors were purchased from Selleckchem (Houston, TX). Trypsin-EDTA, DMEM, RPMI, penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). Cells were purchased from the ATCC and were not further validated beyond that claimed by ATCC. Cells were re-purchased every ~6 months. The plasmid to express GRP78/BiP/HSPA5 was kindly provided to the Dent laboratory by Dr. A.S. Lee (University of Southern California, Los Angeles, CA); all other plasmids were purchased from Addgene. Commercially available validated short hairpin RNA molecules to knock down RNA / protein levels were from Qiagen (Valencia, CA) or were supplied by collaborators. Rabbit antiserum for mouse PERK phosphorylated at T799 was developed as described [44 (link)]. Reagents and performance of experimental procedures were described in refs: [12 (link)/13 (link)/17 (link)/18 (link)/21 (link)–24 (link)/33 (link)/40 (link)].

A kinase inhibitor library containing 273 compounds (L1200) was obtained from SelleckChem (Munich, Germany), with compounds predissolved to 10 mM in DMSO. Analytical-grade DMSO was obtained from Biosolve B.V. Rapamycin, roscovitine, sorafenib tosylate, torin 1, and buparlisib (NVP-BKM-120) were purchased from SelleckChem through distributor Bio-Connect B.V. (Huissen, Netherlands). Metformin HCl was obtained from Sigma-Aldrich.

To broadly inhibit the protein-tyrosine phosphatases, pervanadate is prepared by incubating 10 mM sodium orthovanadate with 0.15% hydrogen peroxide in 20 mM HEPES for 5 min at room temperature. Pervanadate is neutralized with catalase and added to cells immediately. For western blot, cells were lysed 10 mins after pervanadate addition, for live-imaging, cells were imaged immediately after pervanadate addition.
To perturb the actin cytoskeleton, 4 hr post transfection, the media was replaced with complete media supplemented with cytoskeletal drugs CK-666 (Sigma Aldrich), Wiskostatin (Krackeler Scientific) and smifH2 (EMD Millipore) at specified concentrations. DMSO was used as vehicle control. 18 hr post transfection, splitYFP was matured at 30°C. At 24 hr post transfection splitYFP fluorescence was quantified as described above.
To inhibit Raf kinase, 4 hr post transfection, the media was replaced with compete media supplemented with sorafenib tosylate (Selleckchem). DMSO was used as a vehicle control. 18 hr post transfection, splitYFP was matured at 30°C. At 24 hr post transfection splitYFP fluorescence was quantified as described above.