MTT assay

The MTT assay was used to assess the anti-proliferative effects of metformin in MCF-7 and 4T1 cells. In recent years of breast cancer research, the dosage range of metformin has been 1–50 mM, and the highest dosage can reach 100 mM (38,39). It has been reported that metformin can induce the AMPK signaling pathway in the dose range of 10–20 mM, thereby increasing apoptosis (40). Therefore, metformin with the dose range of 20–50 mM was selected to study its antitumor mechanism. Exponentially growing cells were trypsinized, counted and plated at 5×10³ cells/well in 96-well plates. After 24 h, cells were treated with 0, 20 or 50 mM metformin for 24, 48, 96 or 144 h. Thereafter, the medium was changed, and the cells were incubated with 10 µl MTT (Sigma-Aldrich; Merck KGaA; 5 mg/ml) at 37°C for 4 h before each test. The supernatant was then carefully discarded, and 150 µl DMSO (Sigma-Aldrich; Merck KGaA; cat. no. D2650) was added to dissolve the precipitate. Absorbance at 490 nm (A570) for the experimental group and DMSO (control) was measured using a microplate reader (KHB ST-360; Shanghai Kehua Bio-Engineering Co., Ltd.). Finally, actual absorbance was calculated using the following formula: Actual absorbance = absorbance of treated cultures-absorbance of DMSO. To ensure consistency, the experiment was repeated three times in all cases.