*4.3. Cell Proliferation Assays*

Cell viability was analyzed using a standard MTT assay. Fifteen thousand MCF-7 cells or 6000 MDA-MB-231 cells were plated in each well of a 24-well plate in a total volume of 400µL of culture medium. After 4 h of culture, cells were treated with inhibitors, as described above. The cell viability was assessed at 6, 12, 24, 48, and 72 h after treatment. Forty microliters of MTT (5 mg/mL) was added to each well and further incubated for 1 h at 37 ◦C. Wells were left to dry at 37 ◦C, and 400 µL of DMSO (dimethylsulfoxide) was added and left at 37 ◦C for 10 min. One hundred microliters of DMSO from each well was transferred to 96-well plates in triplicates. The absorbance values were read at 570 nm using a Multiskan™ GO microplate spectrophotometer (Thermo Fisher Scientific, Gillingham, UK).

Alternatively, at the given time points after treatment, the cells were harvested, diluted in trypan blue at a ratio of 1:4, viewed using a hemocytometer and microscope, and counted manually.
