Mouse xenograft models

Four- to six-week-old female BALB/c nude mice were obtained from Guangdong Medical Lab Animal Center. To establish the liver tumor xenograft mouse models, 5 × 10⁶ SMMC-7721 cells in 200 μL saline were implanted subcutaneously into the right flank of the nude mice. After implantation, tumor volumes were measured with a slide calliper every 2 days and calculated using the following formula: 0.5 × (length) × (width)². When the tumor size reached 100 mm³, the tumor-bearing mice were divided randomly into two groups and treated with the vehicle (1% DMSO in saline) or 10 mg/kg JNU-144 in saline every 2 days by i.p. injection. The mice were sacrificed after six times injection, and tumor tissues were excised, weighed and photographed. Subsequently, xenograft tumor and the major organs were fixed in neutral buffered formalin for western blot analysis, immunohistochemistry and pathological examination.

All of the animals were treated according to protocols approved by Institutional Animal Care and Use Committee of the Shenzhen People’s Hospital. And this study was approved by Institutional Animal Care and Use Committee of the Shenzhen People’s Hospital (Approval Document No. LL-KT-201701017).