Pf 05175157

The MET (PHR1084, Sigma-Aldrich, Laramie, WY, USA) stock solution was prepared in standard NB cell culture medium. The acetyl CoA carboxylase (ACC) inhibitor PF 05175157 (5790, Tocris, Bristol, UK) was dissolved in DMSO and stored at −80 °C. 1.9 × 10⁴ cells/well of SKNBE(2) cells and 8 × 10³ cells/well of KELLY cells were seeded in 96-well plates. Then, the cells were treated with different concentrations of MET (1 to 20 mM) for 72 h, or with PF 05175157 (3–33 µM) for 48 h. Standard medium was used as a vehicle control of the MET-treated cells, whereas DMSO was used as a vehicle control of the PF 05175157-treated cells. Cell viability was measured via a crystal violet assay, as previously described [28 (link)].

Naive CD4⁺ T cells were isolated from the spleen of female mice at the age of 6–8 weeks with MojoSort Mouse CD4⁺ Naïve T Cell Isolation Kit (Biolegend) according to the manufacturer’s instructions. For Th0 induction, 2 to 3 × 10⁵ naive CD4⁺ T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Biological Industries), penicillin-streptomycin (500 U; PAN biotech), and β-mercaptoethanol (50 μM; Sigma-Aldrich) and stimulated with Dynabeads Mouse T-Activator CD3/CD28 (Gibco) at a cell:bead ratio of 1:1 in the presence of rmIL-2 (200 U/ml; Peprotech) (Wang et al., 2019 (link)). To obtain iTreg cells, rhTGFβ1 (2 or 0.5 ng/ml; Peprotech) was simultaneously added along with the reagents used for inducing Th0 cells. ACLY inhibitor: SB204990 (Tocris Bioscience), ACC inhibitor: PF05175157 (Tocris Bioscience), FASN inhibitor: cerulenin (Biovision), protein synthesis inhibitor: CHX (Tocris Bioscience), proteasome inhibitor: MG132 (Selleck), lysosome inhibitor: leupeptin (Selleck).