Wst 1

Approximately 10,000 VERO cells were cultured in 96-well plates and incubated with 150 μM of kaempferol or 150 μM MTZ for 40, 60, 90, and 180 min; VERO cells in the presence of DMSO and without kaempferol were used as controls. The total volume of each well was adjusted to 200 μL. Briefly, 95 μL of PBS (pH 6.8) + 5 μL of WST-1 (Sigma-Aldrich, MO, USA) was added to 100 μL of WST-1 in each well. The plate was then incubated and protected from light for 30 min at 37 °C. Then, 50 μL of the supernatant was taken from each well, placed in another 96-well plate, and finally read in a microplate reader Synergy HT (Biotek, VER, USA) at a wavelength of 450 nm.
This experiment was repeated three times and the data presented here are an average of the standard values. The percentage viability of VERO cells was determined by inserting the optical densities into the formula: %Viability = [(mean Optical Density (O.D) treated cells × 100]/(mean O.D. control cells) [44 (link)].

Cell Growth Assay. Breast cancer cells growth rates were measured using the WST-1 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1, Sigma-Aldrich, CA, USA) method. First, the cells were seeded in a 96-well plate at 5 × 103 cells/well, incubated at 37 °C for 24 h, transferred to serum-free medium, and transfected with S63A and S262A as described above. Then, after cultivation under 21% O2 for 24 h, the cells were transferred to a culture medium containing 10% FBS. Finally, WST-1 was added to the wells, and the plates were incubated at 37 °C for 3 ~ 4 h for efficient cell dyeing, and analyzed for its absorbance at 460 nm using a spectrophotometer (Molecular Devices, USA). And, cell viability was measured by tryphan blue staining. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue. When a cell suspension is simply mixed with the dye, add equal parts of 0.4% trypan blue dye to the cell suspension to obtain a 1 to 2 dilution, and incubate mixture for less than 3 min at room temperature. Place the hemacytometer with 1:1 tryphan blue staining cells on the stage of a light microscope (Olympus Optical Co., Tokyo, Japan) and calculated the staining cells.

Cell survival was measured by MTT assay with the kit (Cat# 11465007001, Sigma-Aldrich) and following procedures provided by the manufacturer. Briefly, the cells were grown in a 24-well plate. After treatment, 50 µl of MTT reagent was added to each well and incubated for 3 h. The plate was then allowed to stand overnight in the incubator with 500 µl solubilization buffer. Absorbance at 580 and 670 nm was measured on a Bio-Tek plate reader. For WST-1 (cat#11644807001, Sigma-Aldrich) assay, the cells were incubated with 50 µl of WST-1 solution for 2 h in 24-well plates. Cell viability was measured at 450 and 600 nm in a microplate reader. For both MTT and WST-1 assays, data were expressed as mean ± s.e.m. of corresponding controls from three independent experiments, 6 repeats per each treatment of one independent experiment. The SPSS one-way ANOVA was employed to analyze all data with the post hoc Turkey to compare the difference between any two groups.