Verteporfin

Cells were seeded at 2 × 10⁴ cells/ml in MEM/EBSS, supplemented with 10% FBS. The cells were treated with Ro 08-2750 (5 and 10 μM, cat. no.: 2272, TOCRIS, America), dimethyl sulfoxide (DMSO), Verteporfin (1 μM, VP, a YAP inhibitor, catalog no.: HY-B0146, MedChemExpress, China), and 200 ng/ml β-NGF (cat. no.: 450-01, PEPROTECH, US) for 24, 48, and 72 h. After treatment, cell numbers were counted using a hemocytometer. To carry out the colony formation assay, 200 cells/well were plated into 60-mm cell culture dishes, and colonies were counted after 2 or 3 weeks; the cells were fixed with methanol (≥ 99.5%) at room temperature for 15 min and stained with a 1% crystal violet solution at room temperature for 10 min. The NGF inhibitor was added to the cells every week. Only colonies containing more than 50 cells were regarded as positive colonies and counted. All experiments were conducted in triplicate.

The human HCC cell line BEL/FU and the parent cell line BEL-7402 were obtained from KeyGen Biotech Co., Ltd. (Nanjing, China). The BEL/FU cells and BEL-7402 cells were cultured in RPMI-1640 (Biological Industries, Israel) containing 10% foetal bovine serum (Biological Industries, Israel). The HCC-LM3 cell line was provided by the Cell Bank of the Shanghai Institutes of Biological Science, Chinese Academy of Sciences and cultured in DMEM high glucose medium (Biological Industries, Israel) with 10% foetal bovine serum (Biological Industries, Israel). SK-Hep-1 cells were provided by the China Center for Type Culture Collection and cultured in DMEM containing 10% foetal bovine serum at 5% CO₂ and 37 °C. EBSS (Gibco) was used to provide starvation conditions. CQ (Selleck, USA) and 3-MA (Selleck, USA) were used to block autophagy. Verteporfin, 5-Fu, and DOX were purchased from MedChemExpress (MCE, USA).

Insulin-like growth factor-1 (IGF-1) and PI3K inhibitor Wortmannin (WM) were purchased from Sigma-Aldrich (Milan, Italy). The dual insulin-like growth factor 1 receptor (IGF-1R)/insulin receptor (IR) inhibitor OSI-906 (Linsitinib) was obtained from Tocris Bioscience (Space, Milan, Italy). Focal Adhesion Kinase selective inhibitor VS-4718 (PND-1186) was bought from Santa Cruz Biotechnology (DBA, Milan, Italy). The YAP/TEAD complex suppressor Verteporfin was purchased from Med Chem Express (DBA, Milan, Italy). All the aforementioned compounds were dissolved in dimethyl-sulfoxide (DMSO) except IGF-1 which was dissolved in water.

Information of antibodies was documented in Table S1. DOX and verteporfin were from MedChem Express (USA). TUNEL stain kit, CCK-8 kit, DHE and DCF reagents were purchased from Beyotime Institute of Biotechnology (Shanghai, China). ATP assay kits was obtained from Jiancheng Bioengineering Institute (Nanjing, China). ELISA kits for plasma cTnI and Gal-3 determination and lactate assay kit were from Elabscience Biotechnology Co., Ltd (Wuhan, China).

Recombinant Human S100A8/A9 Heterodimer (carrier-free) (#753406) was purchased from Biolegend® (San Diego, CA). RAGE antagonist FPS-ZM1 (#S8185), AKT inhibitor Ipatasertib (#S2808), ROCK inhibitor Y-27632 2HCl (#S1049) and Myosin II inhibitor Blebbistatin (#S7099) were obtained from Selleckchem. FAK inhibitor VS-4718 (# HY-13917) and YAP/TEAD binding disruptor Verteporfin (# HY-B0146) were purchased from MedChemExpress (MCE). rhS100A8/A9 was diluted at 100 μg/mL in 1X PBS, while each pharmacological inhibitor was prepared as a 10 mM stock solution in DMSO.

Nobiletin (>99% purity) was purchased from MedChemExpress (Monmouth Junction, NY, USA); the CCK-8 solution was obtained from Dojindo Molecular Technologies, Inc. (Tokyo, Japan); fetal bovine serum was obtained from ZETA (San Francisco, CA, USA); trypsin and dimethyl sulfoxide (DMSO) were obtained from Thermo Fisher Scientific (Waltham, MA, USA); Eagle’s minimum essential medium (EMEM) and McCoy’s 5A media were obtained from Gibco (Gaithersburg, MD, USA). Antibodies against AKT, STAT3, SRC, YY1AP1, caspase 3, caspase 9, phospho-AKT, phospho-STAT3, phospho-YY1AP1, phospho-SRC, cleaved caspase 3, and cleaved caspase 9, as well as all secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA). Stattic and Verteporfin were obtained from MedChemExpress. IGF1 was obtained from BD Bioscience (Shanghai, China).