Fatostatin

G-1 (HY-107,216), RSL3 (HY-100,218 A), Erastin (HY-15,763), A939572 (HY-50,709), NSC781406 (HY-100,470), and Fatostatin (HY-14,452) were purchased from Med-Chem-Express. All these reagents were dissolved in DMSO, which was obtained from Servicebio (GC203005). The primary antibodies used in the present study included GPER1 (ABclonal, A10217), SCD1 (ABclonal, A16429), SREBP1 (Servicebio, GB113804), PI3K (ZENBIO, R22768), Phospho-PI3K (Tyr467/Tyr199) (ZENBIO, 310,164), Phospho-mTOR (Ser2448) (ZENBIO, 381,557), AKT (ABmart, T55561), Phospho-AKT (Ser473) (ABmart, T40067), GAPDH (ABclonal, AC033), mTOR (ABmart, T55306), GPX4 (ZENBIO, R381958), and NRF2 (ABmart, T55136). The second antibodies, horseradish peroxidase (HRP) goat anti-rabbit IgG (ABclonal, AS014) and (Servicebio, G1213), HRP goat anti-mouse IgG (ABclonal, AS003) were applied. A dilution ratio of 1:1000 was used in western blotting and 1:100 in immunohistochemistry and immunofluorescence analyses.

Cells were seeded at appropriate densities/100 µL/well in 96-well plates in 8 replicates. Detailed densities are listed in Supplementary Table S1. Cells were treated with inhibitors, chemotherapeutics, or in combinational approaches simultaneously at the indicated concentrations for 72 h if not stated otherwise. Cell viability was measured indirectly by fluorescence detection with a Spark^® multimode microplate reader (Tecan, Männedorf, Switzerland) after 2 h of Resazurin treatment (30 µg/mL, R12204; Thermo Fisher Scientific, Waltham, MA, USA). Experiments were performed in triplicates. Graphpad Prism software v8.4.3 (RRID:SCR_002798) was used for the calculation of IC₅₀ values and SynergyFinder (RRID:SCR_019318) was used for the modulation and calculation of synergy scores [26 (link)]. In combinational analyses with inhibitors, stable and non-lethal concentrations (approximately 10% reduction in cell viability or published concentrations) were used: 4 µM GSK126 (HY-13470; MedChemExpress, Sollentuna, Sweden), 4 µM EPZ6438 (HY-13803; MedChemExpress, Sollentuna, Sweden), 125 nM DZNep (Cay-13828; Biomol, Hamburg, Germany), 1 µM Fatostatin (HY-14452; MedChemExpress, Sollentuna, Sweden), or 30 µM Glibenclamide (HY-15206; MedChemExpress, Sollentuna, Sweden) [27 (link)].

RORγ inhibitor XY018 (purity >98%) was obtained from Tocris Bioscience (Bristol, UK). SREBP2 inhibitor fatostatin (purity >99%) and HMGCR inhibitor atorvastatin (purity >98%) were obtained from MedChemExpress (Monmouth Junction, NJ, USA) and Selleck Chemicals (Houston, TX, USA). Cytarabine was obtained from Stadapharm (Bad Vilbel, Germany).

The antiproliferative effect of avasimibe and fatostatin (MedChemExpress, Shanghai, China) was evaluated with a Cell Counting Kit-8 kit (CCK-8, Vazyme, Nanjing, China). One day before avasimibe treatment, GC cells were seeded in 96-well plates (5,000 cells/well). The cells were treated with avasimibe for 24 h or 48 h. After the indicated time, 10 µL per well of CCK-8 solution was added and incubated at 37°C for 1 h. Absorbance was recorded at 450 nm, and five independent assays were carried out.
For the plate colony formation assay, GC cells were seeded in 6-well plates or 12-well plates (1,000 cells/well in 6-well plates and 500 cells/well in 12-well plates) and incubated for 10–14 days. The medium with or without avasimibe was changed every other day. Then, the cells were fixed with 4% paraformaldehyde for 15 min and stained with crystal violet for 1 h. Images were acquired with a digital camera, and three independent assays were carried out.