E0228

The viability of HGC‐27 or SNU‐1 cells was analyzed using an MTT Cell Proliferation and Cytotoxicity Assay Kit (M1020) from Solarbio (China). HGC‐27 or SNU‐1 cells were cultured routinely for 24, 48, or 72 hours in a 96‐well plate, and then, 90 μl of fresh medium and 10 μl of MTT reagent were added to each well to further culture the cells for 4 hours. Afterward, 110 μl of Formazan solution was added and shaken for 10 minutes. Finally, the absorbance was measured at 490 nm using a microplate absorbance reader (E0228) from Beyotime (China).

The concentration of 2‐deoxy‐d‐glucose‐6‐phosphate (2‐DG6P) in LX‐2 cells was measured utilizing 2‐deoxy‐d‐glucose (2‐DG) uptake measurement kit (CRS‐OKP‐PMG‐K01E; Cosmo Bio) to detect the cell glucose uptake following the manufacturer's directions. LX‐2 cells were seeded in a 96‐well plate at a density of 1500 cells/well, differentiated, and then maintained for 4 days before use. The cells were starved for glucose by preincubating with 100 µL of Krebs‐Ringer‐Phosphate‐HEPES buffer (MG6617; MesGen Biotech) containing 2% bovine serum albumin (BSA) for 40 min, and then stimulated or not with 1 μM insulin for 20 min to activate the glucose transporter. Next, cells were treated with 10 µL of 10 mM 2‐DG and incubated for 20 min. According to the manufacturer's specification, appropriate reagent was applied to induce nicotinamide adenine dinucleotide phosphate generation which was the detection index of 2‐DG6P content. The absorbance values were measured at 412 nm utilizing a microplate reader (E0228; Beyotime).

MTT assay (M1020, Solarbio, China) was performed to access cell viability. Breast cancer cells were seeded in 96-well plates at a density of 3 × 10³ per well, and stimulated by various intensities of ultrasonic irradiation. Following incubation, the medium solution was replaced and 10 μl MTT solution was added for another 4-h incubation with cells. After the supernatants were discarded, 110 μl Formazan solution was added to each well and oscillated for 10 minutes (min). The absorbance value at 490 nm was estimated by a microplate reader (E0228, Beyotime, China).