T 25 flasks

Human umbilical vein endothelial cells (HUVECs American Type Culture Collection, Manassas, VA, USA) were cultured in a CO₂ incubator using Medium 199 (M199, LabClinics, Barcelona, Spain) supplemented with penicillin-streptomycin (100 units and 0.1 mg/mL, respectively) and 10% fetal bovine serum (complete M199) in T-25 flasks (SPL Life Sciences Co., Ltd.), allowed to grow up to 70–80% convergence and replated by trypsin treatment. Unspecific toxicity of the samples was tested with the WST-1 cell viability assay (Roche Applied Science, Penzberg, Germany), following the manufacturer’s recommendations. Briefly, HUVECs were seeded in 96-well plates at a density of 5000 cells per well in 100 µL of complete M199. After a 24-h incubation at 37 °C, the medium was removed, and 90 µL of fresh M199 were added together with 10 µL of the sample of interest in PBS. HUVECs were placed back in the incubator for 24 h or 48 h. At the moment of reading, 10 µL of WST-1 reagent was added to each well, and, after an incubation of 3–4 h, absorbance at 440 nm was measured with an Epoch^TM microplate spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). For each sample, three different concentrations were tested in triplicates, and each plate contained three seeded wells with 1% bleach (0% viability control) and three wells with 10 µL PBS (100% viability control) as controls.

Primary NK cells were purified (> 95% pure) from peripheral blood mononuclear cells (PBMCs) of healthy human volunteers (n = 26; female n = 12, male n = 14). This research protocol was reviewed and approved by the institutional review board of CHA Bundang Medical Center, CHA University (permit number: CHAMC 2017–01–001). PBMCs were isolated from whole blood by a density gradient with Ficoll-Paque Plus (GE Health Care, Piscataway, NJ) followed by purification using an NK cell isolation kit (Miltenyi Biotec, Germany). Cells were activated in CellGenix® GMP Stem Cell Growth Medium (SCGM, CellGenix, Freiburg, Germany) supplemented with 10% human serum (Sigma-Aldrich, St. Louis, MO) and the cytokines IL-2 (10 or 100 ng/ml), IL-15 (10 ng/ml), IL-27 (10 ng/ml) (Peperotech, Inc. NJ) and IL-18 (10 ng/ml) (R&D Systems, Inc., MN) under standard culture conditions (a humidified 5% CO₂ atmosphere, 37 °C) for 21 days (long-term culture). Fresh culture medium containing cytokines and 10% human serum was added to the flask every 2 to 3 days for 21 days. The initial seeding density of NK cells was a median of 1 × 10⁶ (range: 0.8–1.3 × 10⁶) NK cells in 6-well plates (SPL Life Sciences, KOR). After 7 days, the cells were transferred into T25 flasks, T75 flasks, and T175 flasks (SPL Life Sciences, KOR) with additional media containing cytokines for 21 days.

MUSE cell analysis assay kit (Millipore, USA) that contains PI and RNase A premixed reagent was used for cell cycle analysis. MDA-MB-231 and MCF-7 cells were seeded at a density of 5 × 10⁵ cells/mL in complete DMEM high glucose (Nacalai, Japan) in T25 flasks (SPL Life Sciences, Korea) and incubated for 24 h at 37°C in a humidified incubator (ESCO, Singapore) with 5% CO₂ and 95% air. Cells were treated with different doses of 3F1: e.g., IC₅₀, lower than IC₅₀, higher than the IC₅₀ to evaluate whether the compound 3F1 consistently affected the cell cycle of these breast cancer cells. Treated and control cells were incubated for 24 h at 37°C in a humidified incubator supplied with 5% CO₂ and 95% air followed by fixation and staining of the cells by manufacturer instructions of the kit. Analysis was done using the MUSE cell analyzer (Merck-Millipore, USA) followed by ModFit LT version 5 to generate the histograms and the percentage of cells in each cell cycle phase.