Sorafenib

The effect of TMEA on the migration of HUVECs was evaluated as previously described (Walter et al., 2015 (link)). After HUVECs were scratched with a sterile pipette tip, the cells were treated with medium containing 0, 3, 15, 30, and 60 μM of TMEA and 10 μM of sorafenib (TargetMol, Boston, MA, USA) for 24 h. Three randomly selected views along the scraped lines were photographed on each well at 0 and 24 h after TMEA treatment under a phase-contrast inverted microscope at a magnification of 40×. The horizontal distance was quantified at 0 and 24 h for the migration ability using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). The migration distance was calculated as follows: migration distance = distance within scratch (0 h) − distance within scratch (24 h).

Atorvastatin was purchased from Cayman Chemicals (Ann Arbor, MI). Tannic acid and sorafenib was purchased from TargetMol (Shanghai, China). Control adenovirus, GFP adenovirus (Ad-GFP), and KLF2 overexpression adenovirus (Ad-KLF2) with a C-terminal Flag/His tag was custom made at Weizhen Biosciences Inc. (Jinan, Shandong, China).

All the experimental procedures rigorously complied with the Guide for the guidance of Animal Ethics Committee of Fudan University. Murine Hepa 1-6 cells (2×10
⁶; National Collection of Authenticated Cell Cultures, Shanghai, China) were resuspended in 100 μL PBS and injected subcutaneously in the right flanks of 6-week-old C57BL/6 mice (Shanghai Jihui Laboratory Animal Care Co., Ltd., Shanghai, China). One day before the inoculation of cancer cells, mice were treated with cobra venom factor (CVF, 0.2 mg/kg, Catalog #ZX0006; Rongmin Biotechnology Center, Shanghai, China), and continued every other day until the end of the experiment. For the inhibitor administration, tumor-bearing mice were treated with sorafenib (10 mg/kg, T0093L; TargetMol, Wellesley Hills, USA), lenvatinib (10 mg/kg, T0520; TargetMol), and BI-2536 (20 mg/kg, T6173; TargetMol) at days 8, 10, and 12 after cell inoculation. Tumor volumes were measured every other day, and the tumor volumes were calculated using the formula: volume=0.5×width× width×length.

Human ovarian cancer SKOV-3 cells were obtained from the Shanghai Institute of Cell Biology (China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) in a CO₂ incubator at 37°C. The cells were passed once every 2–4 days.
There were 5 cell treatment groups (n = 3 per cell treatment group), i.e., SKOV-3 cell control, TGF-β1 (10 ng/mL)-alone treatment according to previous studies [28 (link),29 ,30 (link)], sorafenib (10 µM)-alone, sorafenib (10 µM) + TGF-β1 (10 ng/mL), and TGF-β1 (10 ng/mL) + Ly2157299 (a selective TGF-β1 inhibitor, 5 µM) group. Human TGF-β1 was obtained from Pepro Tech (Shuzhou, China), while sorafenib and Ly2157299 were from TargetMol (Shanghai, China).

For dose-response curves, 2000 MCF10A cells were seeded in 96-well plates and treated with the indicated concentrations of the MAPK (ERK) inhibitor FR180204 (Hölzel Diagnostika) or the RAF1 inhibitor sorafenib (TargetMol) in EGF-depleted medium, and viability was assessed by CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) MTS assay after 48 h.

HeLa cells were selected for routine trypsin digestion, and the cell concentration was adjusted to 2 × 10⁷/ml. Six-week-old male BALB/c nude mice (Beijing Vital River Laboratory Animal Technology, China) were randomly selected and 0.3 ml of cell suspension was injected subcutaneously under the left flank. Sorafenib (T0093L, TargetMol Chemicals, USA) and a control saline were administered after 2 weeks of tumor bearing. And Solafenib was injected intraperitoneally for 14 days (10 mg/kg/day). The body weight and tumor size of nude mice were monitored. The tumor volume was measured by an electronic caliper using the formula as follows: tumor volume (mm³) = length (mm) × width (mm) × height (mm) × π/6. After 14 days of Solafenib administration, mice were euthanized and the tumor tissues were taken for pathological observation and analysis. All test procedures were abided by the Guiding Principles for the Care and Use of Laboratory Animals and reviewed and approved by the Committee on the Ethics of Animal Experiments of China Agricultural University (Beijing, China; Approval Code: AW81103202-4-1).