Mg132 m8699

HCC cell lines SMMC-7721, Huh7, HepG2, Hep3B and 293T cells were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). HCC Cell lines were routinely cultured in Dulbecco's modified Eagle's medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco). Spheres from OV6⁺ HCC cells were cultured in DMEM/F-12 (Gibco) without FBS, but with supplement of ITS Liquid Media (Sigma-Aldrich, St. Louis, MO, USA), B27 (Invitrogen, Carlsbad, CA, USA), EGF Recombinant Human Protein Solution (Gibco), FGF-Basic (AA 1-156). Recombinant CXCL12 protein (R&D Systems, Minneapolis, MN, USA) was added in conditional media for the CXCL12/CXCR4 related assays. AMD3100 (Plerixafor, HY-10046) was purchased from MedChem Express (Monmouth Junction, NJ, USA). MG132 (M8699) and CHX (R750107) were obtained from Sigma-Aldrich.

The following antibodies were employed in western blot: anti-TIM23 (1:1000; BD Biosciences, 611223), anti-LC3B (1:1000; Proteintech, 146000-1-AP), anti-PCNA (1:1000; Proteintech, 10205-2-AP), anti-β-actin (1:5000; Proteintech, 60008-1-1g) and anti-HIF1α (1:1000; Abclonal, A11945). Anti-p-18-FUNDC1 (1:500) and anti-FUNDC1 (1: 1000) polyclonal antibodies were produced by immunizing rabbits with synthesized, purified phosphorylated and non-phosphorylated peptides from FUNDC1 (Abgent, SuZhou, China). For immunofluorescence, the following antibodies were used: anti-Ki67 (1:100; Affinity, AF0198) and anti-α-SMA (1:200; Santa Cruz, sc-32251). The secondary antibodies used for immunofluorescence were: goat anti-mouse IgG Alexa Fluor-488 (1:200; Molecular Probes, A11029) and goat anti-rabbit IgG Alexa Fluor-561 (1:200; Molecular Probes, A11008). The cell-penetrating peptides P (NH2-GRKKRRQRRRPQDYESDDESYEVLDLTEY-COOH) and C (NH2-GRKKRRQRRRPQDYESDDESpYEVLDLTEY-COOH) were synthesized by Nanjing Jietai Company (Nanjing, China). All the above peptides were HPLC purified and had a purity of greater than 98%. 2’,7’-Dichlorofluorescin diacetate (DCFH-DA) was provided from Invitrogen (D2938), N-Acetyl-L-cysteine (NAC) from Beyotime Institute of Biotechnology (ST1546), chloroquine (C6628) and MG132 (M8699) from Sigma-Aldrich.

Regorafenib (Regora; R-0443565) was provided by Heowns (Tianjin, China). Chloroquine (CQ; C6628) and MG132 (M8699) were purchased from Sigma-Aldrich (Shanghai, China). Z-VAD-FMK (C1202) was purchased from Beyotime (Shanghai, China). ALW-II-41-27 (HY-18007), cycloheximide (CHX; HY-12320), TBHQ (HY-100489) and EFNA1 recombinant protein (HY-P72662) were provided by MedChemExpress (New Jersey, USA). Schisandrin C (Schi C; FY1671) was provided by Feiyubio (Nantong, Jiangsu, China). Ponatinib (Pona; T2372), dasatinib (Dasa; T1448), nilotinib (Nilo; T1524), bosutinib (Bosu; T0152) and crizotinib (Crizo; T1661) were purchased from Topscience (Shanghai, China). Antibodies used for western blot, immunofluorescence and immunohistochemistry analyses are showed in Supplementary Table 2. FDA-approved drugs and natural products for drug screening were purchased from Topscience (Shanghai, China) and Nantong Jingwei Biotechnology Co., Ltd (Nantong, Jiangsu, China), respectively. Related information was shown in Supplementary Table 3.

Cells were seeded in 96-well plates or in 12-well plates coverslips and were left in the incubator untreated or exposed to DMSO or drug treatment: O-glycosylation inhibitor Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (B4894) for 48 h or proteasome inhibitor MG132 (M8699) for 1 h, both purchased from Sigma-Aldrich (St. Louis, MO, USA). After fixation with paraformaldehyde, coverslips were permeabilized with PBS 0.2% Triton X-100 (Merck Millipore, Burlington, MA, USA). Nonspecific binding was blocked with 10% BSA in PBS for 30 min at room temperature. Cells were stained with primary antibodies overnight at 4 °C. Antibody concentrations: CD44 at 1:400; CD44v9 at 1:100; CD44v at 1:200; Tn, STn, and T undiluted. Secondary antibodies anti-mouse and anti-rat Alexa Fluor^® 488 and anti-mouse Alexa Fluor^® 594 were used at 1:500. Nuclear stain was performed with DAPI (Sigma-Aldrich). Cells were washed with PBS, and images were acquired using Zeiss AxioImager Z1 (Carl Zeiss, Oberkochen, Germany) or IN Cell Analyzer 200 (GE Healthcare, Little Chalfont, UK).

PGE₂ (14,010), PGE₁-alcohol (13,020), and ONO-AE3-208 (14,522) were purchased from Cayman Chemical (Ann Arbor, MI, USA). OVA (A5503), LPS (L2880), and MG132 (M8699) were from Sigma (St. Louis, MO). MK2206 (S1078), Pioglitazone (S2590) and T0070907 (S2871) were from Selleck (Houston, TX, USA). The average 50% inhibition concentration (IC₅₀) of T0070907 was determined as 1 nM.

Monoclonal mouse anti-HA (H3663), anti-Myc (SAB1305535), and anti-Flag (F3040) antibodies were purchased from Sigma-Aldrich (USA). Polyclonal rabbit anti-IκBα, p-IκBα, P65, p-P65, IRF3, p-IRF3, TBK1, and p-TBK1 were purchased from Cell Signaling Technology (USA). GAPDH, anti-mouse IgG, and anti-rabbit IgG were obtained from Thermo Scientific. Antibodies against PLAU, PLAUR and VISA were purchased from Proteintech. CoraLite594-conjugated goat anti-mouse IgG (H + L) and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (H + L) antibodies were purchased from CST. 3-Methyladenine (3-MA) (Catalogue #189490) and MG132 (M8699) were purchased from Sigma-Aldrich. The Plasminogen Activator/Urokinase enzyme-linked immunosorbent assay (ELISA) KIT was purchased from Solarbio.