Complete dmem high glucose

MDA-MB-231 and MCF-7 cells were seeded at a density of 5 × 10⁵ cells/mL with complete DMEM high glucose (Nacalai, Japan) in T25 flasks and incubated for 24 h at 37°C in a humidified incubator (CCL-170B-8, ESCO, Singapore) with 5% CO₂ and 95% air. Cells were treated with 0.1% DMSO (control) or with the IC₅₀ dose of the 3F1 compound. Single-cell suspension at the recommended density between 1 × 10⁵ and 5 × 10⁶ cells/mL in 1X assay buffer BA was prepared. Staining the cells with DNA-binding dye coupled with DEVD peptide substrate was performed according to the manufacturer's instructions [23 (link), 24 (link)]. The results were analyzed using MUSE analysis software version 1.5. Stained cells were distinguished into four quadrants in a scattered plot.

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.