For the AKT kinase assay, the ADP-Glo? Kinase Assay Kit is used. Active AKT1 or AKT2 kinase and inactive GSK-3β as substrate are mixed by 1× reaction buffer and then added to a white 96-well plate. Pure ATP provided in the kit is serially diluted obtain a final concentration of 0, 1, 10, 50, and 100 μM. GSK-3β is added to reach a final concentration of 2.5, 5, 10 or 20 μM and DMSO is used as a control. The mixed solution is incubated at room temperature and luciferase activity is measured using the Luminoskan Ascent plate reader^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cells are seeded (6×10³ cells/well for KYSE70; 2.5×10³ cells/well for KYSE410; 2×10³ cells/well for KYSE450) in 96-well plates and incubated for 24 h and then treated with different amounts of Oridonin or vehicle. After incubation for 24, 48 or 72 h, cell proliferation is measured by the MTT assay. For anchorage-independent cell growth assessment, cells (2.5, 5 or 10 μM Oridonin) suspended in complete medium are added to 0.3% agar with vehicle, 2.5, 5 or 10 μM Oridonin in a top layer over a base layer of 0.5% agar with vehicle, 2.5, 5 or 10 μM Oridonin. The cultures are maintained at 37°C in a 5% CO₂ incubator for 3 weeks and then colonies are visualized under a microscope and counted using the Image-Pro Plus software program^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[2]
Breast cancer cells are harvested and resuspended in 40% Matrigel-Basement Membrane Matrix, LDEV-free, and then injected (100 μL per site) into the fourth pair of mammary fat pads of nude mice (CrTac: NCr-Foxn1nu). Tumors are measured in two dimensions using manual calipers. Tumor volume is calculated using the formula: Volume = 0.5 × length × width × width. Tumor volume is measured every 2-3 days. Upon harvesting, tumors are fixed in formalin overnight and then in 70% ethanol for histopathology analysis. Mice are treated with Oridonin (15 mg/kg) in 1% Pluronic F68 or vehicle (1% Pluronic F68) daily by intraperitoneal (IP) injection. BEZ235 is reconstituted 1:9 in 1-methyl-2 pyrolidone and polyethylene glycol 300 (PEG300) Mice are treated with this compound formulation at 45 mg/kg daily (QD) by oral gavage^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Song M, et al. Targeting AKT with oridonin inhibits growth of esophageal squamous cell carcinoma in vitro and patient derived xenografts in vivo. Mol Cancer Ther. 2018 Apr 25. pii: molcanther.0823.2017.  [Content Brief]

  [2]. Sun B, et al. Oridonin inhibits aberrant AKT activation in breast cancer. Oncotarget. 2018 Feb 1;9(35):23878-23889.  [Content Brief]