F8758

Panc-1 cells were treated with different concentration (1×, 20×) mixtures of methyl donors based on a previous study by Park et al. [55 (link)]. Basal concentration (1×) was: 17 mg/L L-methionine, 9 mg/L choline chloride, 3 mg/L folic acid, and 2 mg/L vitamin B12, while the 20× concentration was 20 times more of the 1×. L-methionine, choline chloride, folic acid, and vitamin B12 were purchased from Sigma Aldrich (M5308, C7527, F8758, V6629, respectively; St. Louis, MO, USA).

l-methionine, choline chloride, folic acid, and vitamin B12 were purchased from Sigma Aldrich (M5308, C7527, F8758, V6629, respectively; St. Louis, MO, USA). Cells were grown in culture media until 50% confluence, then were treated with different concentrations (×1, ×10, ×20) of the mixture of methyl-donors. Basal concentration (×1) was: 17 mg/L l-methionine, 9 mg/L choline chloride, 3 mg/L folic acid, and 2 mg/L vitamin B12. These concentrations were used according to a previous study of Park et al. [32 (link)].

THP-1 cells and human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC, Manassas, USA). THP-1 cells were cultured in RPMI 1640 medium (11875093, Gibco, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, 10099141, Gibco) and 1% (v/v) PS (100 IU/mL penicillin G & 100 IU/mL streptomycin, C0222, Beyotime, Shanghai, China). HUVECs were cultured in DMEM high glucose medium (11965092, Gibco) supplemented with 10% (v/v) FBS and 1% (v/v) PS. CD14 + primary monocytes were obtained from peripheral blood through magnetic bead sorting and were cultured in RPMI 1640 medium supplemented with 15% (v/v) FBS and 1% (v/v) PS. All cells were maintained in a humidified atmosphere containing 5% CO₂ at 37 °C.
THP-1 cells (8 × 10⁵ cells/mL) in the treatment group were exposed to 2 mL of medium containing 50 mg/L ox-LDL (YB-002, Yiyuan Biotech, Guangzhou, China); 50 μmol/L Hcy (H4628, Sigma-Aldrich, Darmstadt, Germany); 0.1, 0.5, or 1.0 μmol/L FA (F8758, Sigma-Aldrich); 50 mg/L ox-LDL plus 0.5 μmol/L FA; or 50 μmol/L Hcy plus 0.5 μmol/L FA for 72 h. (The medium and induction reagents were changed every 24 h.) CD14 + primary monocytes (6 × 10⁵ cells/mL, 2 mL) were induced and cultured for 12 h using the same grouping method. Cells that were cultured in RPMI 1640 medium without induction reagents were used as a negative control.

Human cells, HT24 (bladder cancer, HTB-4, ATCC), J82 (bladder cancer, HTB-1, ATCC), BFTC909 (colorectal cancer, 60069, BCRC), H292 (lung cancer, CRL-1848, ATCC), A549 (lung cancer, CCL-185, ATCC) and NK-92MI (lymphoma, CRL-2408, ATCC) were obtained from the American Type Culture Collection (ATCC) or Bioresource Collection and Research Center (BCRC). Cells were cultured in DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% (volume/volume; v/v) fetal bovine serum (FBS), sodium bicarbonate and 1% (v/v) antibiotic-antimycotic (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in an incubator with a humidified atmosphere of 5% CO2. NK-92MI cells were cultured in alpha minimum essential medium (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis MO, USA), 2 mM L-glutamine (Thermo Fisher Scientific), 1.5 g/L sodium bicarbonate (Thermo Fisher Scientific), 0.2 mM inositol; (I7508, Sigma-Aldrich), 0.1 mM 2-mercaptoethanol; (M6250, Sigma-Aldrich), 0.02 mM folic acid; (F8758, Sigma-Aldrich), 12.5% horse serum. Latrunculin B (L5288, Sigma-Aldrich). Antibodies, CD161/NK1.1 was from Novus Biologicals (NB100-77528SS, Littleton, CO, USA), vimentin was from abcam (ab92547, Cambridge, UK). Phalloidin staining reagent was from Abcam (ab112125, Cambridge, UK).

The immortalized normal human epidermal melanocyte cell line PIG1 (a gift from Dr. Caroline Le Poole, Loyola University, Chicago, USA) was maintained in Medium 254 (M254500, Gibco, USA), supplemented with human melanocyte growth supplement (Gibco) and 5% fetal bovine serum at 37°C amid 5% CO₂. Oxidative stress was induced by adding H₂O₂ (7722841, Sigma-Aldrich, USA) at 800 or 500 μM into the culture medium for 48 h. To determine the protective effect of FA (F8758, Sigma-Aldrich, USA) on melanocytes against oxidative damages, FA and H₂O₂ were added simultaneously into the culture medium of PIG1 cells at indicated concentrations.