4t1 mouse breast cancer cells

4T1 mouse breast cancer cells were obtained from the American Type Culture Collection (ATCC). The culture medium was Roswell Park Memorial Institute (RPMI) 1640 containing 1% Antibiotic-Antimycotic and 10% FBS. MD Anderson-Metastatic Breast-231 (MDA-MB-231) cells (ATCC) were cultured with a mixture of Ham’s F-12 Medium and Dulbecco’s Modified Eagle’s Medium (DMEM) (1:1) containing 1% Antibiotic-Antimycotic and 10% FBS. Cells were cultured in a humidified incubator with 5% CO₂ at 37 °C.
To test cytotoxicity, cells were seeded into a 96-well plate (5000 cells/well) and incubated overnight to allow cells to attach to the plate. Then, different test agents with various concentrations were diluted with cell culture medium and added into wells (100 μL/well). At the end of treatment, cytotoxicity was determined with 3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay [12 (link),14 (link)–17 (link)]. The absorbance was determined at 570 nm and subtracted by the reference absorbance at 670 nm. The following equation was used to calculate the cell viability:
$$\text{Cell}\text{Viability}\left(\text{\%}\right)=\left({\text{A}}_{\text{Test}}{\text{/A}}_{\text{control}}\right)\times \mathrm{100\%}$$
GraphPad Prism software was used to calculate the IC₅₀.

CHO-PNAd cells were generated as described previously[40 (link)]. 4T1 mouse breast cancer cells were purchased from American Type Culture Collection (VA, USA). Panc02 mouse pancreatic cancer cells were provided by Dr. Claudia Gravekamp, Albert Einstein College of Medicine (New York, NY). CHO-PNAd cells were cultured in α-MEM with 10% FBS and 1% penicillin/streptomycin (pen/strep). 4T1 cells were cultured in RPMI-1640 medium with 10% FBS and 1% pen/strep. Panc02 cells were cultured in McCoy’s medium with 10% FBS, glutamine (2 mM), non-essential amino acids, sodium pyruvate (1 mM), HEPES (10 mM), and pen/strep (100 U/ml). Cells were maintained in a 37°C incubator at 5% CO₂.