96 well culture dishes

Cell viability was measured using a 3-[4,5-dimethylthiazol-2-thiazolyl]-2,5-diphenyl-tetrazolium bromide (MTT) assay that is based on mitochondrial conversion of MTT from a soluble tetrazolium salt into an insoluble colored formazan precipitate, which was dissolved in DMSO and quantified by spectrophotometry (Thermo Multiskan MK3; Thermo Labsystems, Shanghai, China) to obtain optical density (OD) values. OS cells were plated in 96-well culture dishes (Costar, Cambridge, MA, USA) at a density of 1000–2000 cells/well in 100 μL of medium. Serial dilutions were generated from a stock solution of MLN4924 to the desired concentrations. All experimental concentrations were replicated in triplicate. Four hours before the desired time points, 10 μL of 10 mg/mL MTT was added. After a 4h incubation, all media was removed from wells, and 100 μL of DMSO was added. Absorbance percentages for treated cells relative to those of untreated control samples were plotted as a function of drug concentration (log scale). Inhibition of cell viability was measured by the percentage of viable cells relative to the control: % inhibition = 100% × ODT / ODC, where ODT is the average OD value of the treated samples and ODC is the average OD value of the control samples.

MCF-7 and MC3T3-E1 cells were suspended in DMEM and α-MEM culture media and plated at a density of 5.0×10³ cells/well in 96-well culture dishes (Costar, Cambridge, MA, USA). Following 24 h of culture, the medium was replaced with complete culture medium supplemented with various concentrations of drugs. To assess the effects of mSGLXD and RD alone on MCF-7 cell proliferation, MCF-7 cells were treated with mSGLXD (1.25–50 mg/ml) or RD (1.25–50 mg/ml). To assess the effects of mSGLXD and anastrozole alone or in combination on MC3T3-E1 cell proliferation, MC3T3-E1 cells were treated with mSGLXD (0.625–10 mg/ml), anastrozole (0.01–100 μmol/l) or mSGLXD (0.625–10 mg/ml) as well as 10 or 100 μmol/l anastrozole. Following 48 h of drug treatment, the cells were incubated with cell counting kit-8 solution (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) for 2 h. Subsequently, the absorbance (optical density, OD) at 450 nm was measured using a microplate reader (Model 680; Bio-Rad Laboratories, Hercules, CA, USA) and cell viability was calculated according to the following formula: (OD_sample−OD_blank)/(OD_control−OD_blank)×100%.

Cell viability was measured using a Cell Counting Kit-8 (Sigma-Aldrich Shanghai, Shanghai, China) to obtain optical density (OD) values. OS cells were plated in 96-well culture dishes (Costar, Cambridge, MA, USA) at a density of 3000–4000 cells/well in 100 μL of medium. Serial dilutions were generated from a stock solution of compounds to the desired concentrations. All experimental concentrations were replicated in triplicate. Before the desired time points, 10 μL CCK-8 was added. After 2–4 h incubation, the absorbance was measured at 450 nm using a microplate reader. Absorbance percentages for treated cells relative to those of untreated control samples were plotted as a function of drug concentration. Inhibition of cell viability was measured by the percentage of viable cells relative to the control: % inhibition = 100% × ODT/ODC, where ODT is the average OD value of the treated samples and ODC is the average OD value of the control samples.