Dimethylsulfoxide dmso

MLE-12 cells were treated with MG132 (10 μM) (MedChemExpress Co., Ltd., Monmouth Junction, NJ, USA) for 2 h, with dimethylsulfoxide (DMSO) (MedChemExpress Co., Ltd.) as the control [29 (link)]. Lung tissues or cells in different groups were lysed using RIPA buffer, heated at 100℃ for 10 min, and centrifuged at 12,000 g for 10 min. followed by determination of protein concentrations. Lysates were incubated with antibodies (FOXM1: ab207298, Abcam; IgG: ab133470, Abcam) (antibody/lysate = 1 μg/mg), followed by the addition of 30 μL protein A/G plus-agarose beads and overnight incubation at 4 ℃. Samples were added with 25–30 μL protein loading buffer and placed in metal bath at 100 ℃ for 5 min for degeneration. The ubiquitination level of FOXM1 was examined using an anti-Ubiquitin antibody (ab19247, Abcam). Each experiment was performed in triplicate.

A mouse model of bone destruction was prepared by inoculating 5-week-old female C57BL mice under general anesthesia (intraperitoneal (IP) injection of 0.15 mg/kg of medetomidine, 2.0 mg/kg of midazolam and 2.5 mg/kg of butorphanol) with cell suspensions of B16 mouse melanoma cells (1 × 10⁵/10 µL of phosphate-buffered saline [PBS]) via injection into the bone marrow space of the right tibial metaphysis. The mice were then randomly assigned to two groups (n = 8/group). After the B16 cell inoculation, the mice were intraperitoneally administered GANT61 (HY-13901, MedChemExpress, Monmouth Junction, NJ) (40 mg/kg) or dimethyl sulfoxide (DMSO) (cat. no. 046-21981, Fujifilm Wako Pure Chemical Industries, Osaka, Japan) as a vehicle on alternating days from day 3 to day 15. On day 16, all of the mice were euthanized with 150 mg/kg of pentobarbital via IP administration, and the tibiae were removed. The protocols for the mice were approved by the Ethics Review Committee for Animal Experimentation of the Health Sciences University of Hokkaido, Graduate School of Dentistry and Pharmaceutical Sciences (ethical permission code: 19-088). The study was carried out in compliance with the ARRIVE guidelines.