Mg132

The following chemicals were used: DMSO (Sigma-Aldrich, St Louis, MO, Cat# D2650), MG-132 (Cayman Chemical, Ann Arbor, MI, Cat# 10012628), Lumacaftor (Cayman Chemical, Cat# 22196), Tezacaftor (Selleck Chemicals, Houston, TX, Cat# S7059), Elexacaftor (Selleck Chemicals, Cat# S8851), Ivacaftor (Chemscene LLC, Monmouth Junction, NJ, Cat# CS-0497), cycloheximide (CHX, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, Cat# 3720991), doxycycline (dox, FU-JIFILM Wako Pure Chemical Corporation, Cat# 049-31121), glycerol (FU-JIFILM Wako Pure Chemical Corporation, Cat# 075-00616), cyclosporin A (CLP-A, FU-JIFILM Wako Pure Chemical Corporation, Cat# 031-24931), E-4031 (Selleck Chemicals, Cat# S6627). Hit compounds from the in silico screening and the FR3 analog compounds are listed in Supplementary Table S1.

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).

The liver cancer cell lines SMMC-7721, HepG2, Bel-7402, Bel-7404, Huh7, and SK-Hep1 and hepatocyte lines THLE-3 and HL-7702 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All the cells were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% FBS (GIBCO, Carlsbad, CA, USA) and penicillin/streptomycin (GIBCO). Cells were treated with Cycloheximide (CHX, Sigma, St Louis, MO, USA) at a final concentration of 0.1mg/ml and MG132 (Cayman Chemical Co., Ann Arbor, MI) at a final concentration of 25 µM. NMT1- and NMT2-exprssing plasmids were purchased from Origene (Beijing, China). The plasmids expressing NMT1-sh1, NMT1-sh2, POTEE-sh1, POTEE-sh2, RPL7A-sh1, RPL7A-sh2, HBB-sh1, HBB-sh2, HIST1H4H-sh1, and HIST1H4H were purchased from Genechem (Shanghai, China). The expressing plasmids of LXN-HA, FAU-HA, RPL29-HA, LXN-Mut-HA, FAU-Mut-HA, RPL29-Mut-HA, AHSG-HA, ALB-HA, TF-HA, AHSG-Mut-HA, ALB-Mut-HA, TF-Mut-HA, POTEE-Myc, RPL7A-FLAG, HBB-FLAG, HIST1H4H-HA, and HIST1H4H-Mut-HA were purchased from Biolink (Shanghai, China).

Cells lines were cultured in DMEM (Gibco) containing 8% heat-inactivated FBS (Hyclone) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in 5% CO₂. 24 or 48 hours before synchronization, transfection or treatment, cells were seeded on either 6-well Costar plates or 9 cm Falcon dishes. For time-lapse fluorescence microscopy, cells were seeded onto 3.5 mm glass-bottom dishes (Wilco Wells) or 4- to 8-well glass-bottom dishes (Labtek II). For enrichment of cells in mitosis, cells were treated for 24 hours with thymidine (2.5 mM final concentration [Sigma-Aldrich]), released into fresh medium and subsequently treated with nocodazole (415 nM final concentration [Sigma-Aldrich]) for 16 hours. For alignment assays, cells were released from the nocodazole block and cultured in medium containing MG132 (#13697, 5 μM final concentration [Cayman Chemicals]) to block the cells in metaphase.
Other drugs in this study are used as indicated: RO-3306 (#217699 [Calbiochem]); Mps1 inhibitor reversine (#10004412, 50 nM final concentration [Cayman Chemicals]).

The HCC cell lines Bel-7402 and SMMC-7721 and Hepatocyte line HL-7702 cells were cultured in DMEM. The cells were treated with Doxorubicin (Doxo, 0.5-2.0 μg/ml, Sigma, St. Louis, MO, USA), Actinomycin D (10 μg/ml, Beyotime, Haimen, China), MG132 (25 μM, Cayman, Ann Arbor, MI, USA), Wortmannin (50 μM, Cayman) or cycloheximide (CHX, 50 μg/ml, Sigma) for 2-24 h before harvest. shRNAs against Madcam1 (sh#1 and sh#2) and AKT (sh#1 and sh#2) were purchased from Genechem Biotech LTD (Shanghai, China). The cDNA fragments encoding human Madcam1 were purchased from Origene (Beijing, China) and subcloned into the pcDNA3.1 (+) vector with a C-terminal FLAG tag or into the pLJM-based lentiviral vector with a C-terminal Myc tag. The AKT-Myc expression plasmid was described in our previous study [23 (link)]. The primers used for plasmids construction are listed in Supplementary Table.1.

Tween 20, paraformaldehyde, DAPI (4',6-diamidino-2-phenylindole), sulforhodamine B (SRB), cycloheximide (CHX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Camptothecin (CPT) and perifosine were obtained from Calbiochem (Darmstadt, Germany) and AdooQ Bioscience LLC (Irvine, CA, USA), respectively. MG132, epoxomicin and lactacystin were supplied by Cayman Chemicals (Ann Arbor, MI, USA). Chemicals were dissolved in DMSO or water at 20 mM concentrations. Rabbit polyclonal antibodies against HA, c-Myc and PARP-1, and mouse monoclonal antibody against luciferase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal β-tubulin antibody was from Abcam (Cambridge, MA, USA). Mouse monoclonal anti-FLAG (M2) and rabbit polyclonal CHIP antibodies were obtained from Sigma-Aldrich. Rabbit polyclonal GAPDH and β-actin antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from AbFrontier (Seoul, Korea). Rabbit polyclonal HSP90 antibody was purchased from Enzo Life Sciences (Farmingdale, NY, USA). FITC or rhodamine B-conjugated secondary antibody and agarose were obtained from Santa Cruz Biotechnology. Anti-FLAG M2 affinity gel and anti-HA agarose were obtained from Sigma-Aldrich.