Lenvatinib

The PDGFRA mutant and isogenic control clones were seeded into 96-well plates (5000 cells/well) and allowed to adhere overnight. Cell Counting Kit-8 (WST-8, Dojindo, Kumamoto, Japan) was used to assess cell viability/proliferation. To assess drug sensitivity, culture media containing drugs at concentrations ranging from 0.5 to 8 μM was added to cells the day after seeding. Cell viability was evaluated using the WST-8 kit 4 d after treatment. The drugs used were as follows: temozolomide (Tokyo Chemical Industry, Tokyo, Japan), lenvatinib (Cayman Chemical, Ann Arbor, MI, USA), crenolanib (Abcam, Cambridge, UK), abemaciclib, and palbociclib (LKT Laboratories, St. Paul, MN, USA).

The HCC cell lines SNU398, SNU387, Huh7, HepG2, and PLC/PRF/5 were obtained from ATCC. Cells were grown in Roswell Park Memorial Institute (RPMI) medium or Dulbecco’s Modified Eagle’s Medium (DMEM); supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin; and maintained at 37°C with a 5% CO2 atmosphere. Regorafenib (#CS-1205) and sorafenib (#CS-0164) were purchased from ChemScene. Lenvatinib (#19375) and cabozantinib (#18464) were purchased from Cayman Chemical. Trametinib (#S-2673) was purchased from Selleck Chemicals, and buparlisib (#HY-70063) was from MedChem Express. Recombinant human IFNγ (#300-02) was obtained from PeproTech. Cell viability was quantified with Cell Count Reagent SF (nacalai tesque). Absorbance at 450 nm was measured on a micro- plate reader. For quantification of cytotoxicity, we used LDH Cytotoxicity Assay Kit (nacalai tesque). The absorbance value at 490 nm was measured.

Sorafenib, regorafenib and lenvatinib were from Cayman Chemical Company (Michigan, United States); DMSO and bicinchoninic acid (BCA) assay were from Solarbio Science and Technology Company (Beijing, China); fetal bovine serum (FBS), Dulbecco’s modified essential media (DMEM) and trypsin-EDTA were from Gibco (California, United States); cell counting kit-8 (CCK-8) was from Dojindo Corporation (Shanghai, China); penicillin-streptomycin was from MedChem Express (New Jersey, United States); dithiothreitol (DTT) and Tris were from BBI Life Sciences (Shanghai, China); rLys-C and trypsin were from Promega Corporation (Madison, United States); triethylammonium bicarbonate (TEAB) buffer, iodoacetamide (IAM) and bovine serum albumin (BSA) were from Sigma-Aldrich Corporation (St Louis, MO, United States); Annexin V-FITC apoptosis detection kit was from Becton, Dickinson and Company (New Jersey, United States); cell cycle analysis kit was from Beyotime Biotechnology (Shanghai, China); acetonitrile (ACN), formic acid (FA) and TMT 6-plex reagent kit were from Thermo Fisher Scientific (Waltham, MA, United States); protease inhibitor cocktail was from Bimake (Houston, Texas, United States).