Cay10576

The human HCC cell lines PLC/PRF/5 and HepG2 were obtained from American Type Culture Collection (Rockville, MD). Pin1-knockout cell line Pin1^−/− and wild-type Pin1^+/+ cell line were a generous gift from Dr. Zi-Gang Dong (The Hormel Institute, University of Minnesota, MN) [19 (link)]. Pin1^−/− cells, derived from mouse embryo fibroblasts (MEFs), were originally generated from Pin1 knockout mice [20 (link),21 (link)]. PLC/PRF/5, immortalized non-tumorigenic human hepatocyte cell line MIHA, Pin1^−/− and Pin1^+/+ were maintained in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA), and HepG2 was maintained in Minimum Essential Medium (Invitrogen, Carlsbad, CA). CAY10576, MG132, HDAC3 and HDAC4 antibodies were purchased from Cayman Chemical (Ann Arbor, MI). NF-κB inhibitory peptide SN50 was form Calbiochem (Darmstadt, Germany). Antibodies against ZBP-89, p65 and Pin1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-HDAC3 (Ser424) and phospho-IκBα (Ser32/36) antibodies was from Cell Signaling Technology (Beverly, MA). Ad-ZBP-89 viral vector was a generous gift from Dr. JL Merchant (University of Michigan, MI).

HFFF2 recipient cells were treated with several inhibitors at the below concentrations simultaneously with sEV in medium supplemented with 10% (v/v) EV‐depleted FBS and 1% (v/v) A/A for 72 h. Treatment with inhibitors and sEV was repeated 72 h later and readout was determined after 72 h.
Small molecule inhibitor concentrations used for the screen: 40 µM PD98059 (targeting MEK1/2; ThermoFisher), 20 µM SB202190 (p38MAPK; Santa Cruz Biotech), 4µ M TGFB‐R1 (TGFBR1 kinase; Calbiochem), 8 µM VEGFR2 (VEGFR2; Calbiochem), 150 nM GSK429286A (ROCK1/2, Rho‐associated kinase; Abcam), 50 nM CPD22 (ILK, integrin‐linked kinase; Calbiochem), 1 µM CPG (MNK1/2; Calbiochem), 100 nM TORIN2 (mTOR, mammalian target of rapamycin; CAYMAN Chemical), 1µM RUXOLITINIB (JAK1/2 inhibitor; CAYMAN Chemical), 40 µM AG‐490 (JAK2/3 kinase; CAYMAN Chemical), 45 µM JANEX1 (JAK3 kinase; CAYMAN Chemical), 1 µM AG‐879 (protein Tyrosine Kinase; CAYMAN Chemical), 2 µM IMATINIB (tyrosine kinase; TK1; CAYMAN Chemical), 20 µM CAY10576 (IKKε, IκB kinase episilon; CAYMAN Chemical), 1.5 µM SUNITINIB (multi tyrosine kinase, multi‐TK; CAYMAN Chemical). Additional small molecular inhibitors used: 10 µM MLN120B (IKKβ, IκB kinase beta; MedChemExpress) and 10 µM BAY11‐7082 (IKKα/β, IκB kinase alpha/beta; Sigma‐Aldrich). Other pharmacological products: lymphotoxin‐β (Sigma Aldrich‐T7799) and Cisplatin (PHR1624; Sigma‐Aldrich).