The MTT assay is used to evaluate the overall functional integrity and viability of the cultured cells. The MCF-7 cells are put into 96-well plates (2×10⁴ for each well), which are incubated for 24 h. The specific concentrations of alpha-Amanitin and β-Amanitin are added to the cell culture medium, and plates are incubated for an additional 36 h. MTT solution (1:10 ratio) and dimethyl sulfoxide (DMSO) (100 µL) are then added to the cell culture medium and plates are incubated overnight. The absorbance is measured at 570 nm on a plate reader. This experiment is repeated 3 times. The absorbance data are calculated as percentages according to the control group.

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

For tumorigenicity tests, six colonies (untreated) and DTCs derived from MKN45 cells are individually injected subcutaneously into the left and right side of the backs of six 6-week-old female nude mice (BALB/cAjcl-nu/nu). These mice are monitored for 49 days after the inoculation or until tumors reach 10 mm in the largest diameter, and are then euthanized. For the PC model, 1.0×10⁶ MKN45 cells are injected intraperitoneally into six 6-week-old female nude mice (BALB/cAjcl-nu/nu). Mice are then treated with CIS (4.0 mg/kg, intraperitoneal administration) or a combination of CIS and alpha-Amanitin (0.4 mg/kg, intraperitoneal administration). For the combination treatment, alpha-Amanitin is given 24 hours before CIS. Body weight is monitored for 28 days after the treatment.

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

• [1]. Zhao J, et al. Pathological effects of the mushroom toxin alpha-amanitin on BALB/c mice. Peptides. 2006 Dec;27(12):3047-3052.

  [2]. Kume K, et al. α-Amanitin Restrains Cancer Relapse from Drug-Tolerant Cell Subpopulations via TAF15. Sci Rep. 2016 May 16;6:25895.

  [3]. Kaya E, et al. Evaluation and comparison of alpha- and beta-amanitin toxicity on MCF-7 cell line. Turk J Med Sci. 2014;44(5):728-32.

  [4]. Park MW, et al. RNA Polymerase II Inhibitor, α-Amanitin, Affects Gene Expression for Gap Junctions and Metabolic Capabilities of Cumulus Cells, but Not Oocyte, during in vitro Mouse Oocyte Maturation. Dev Reprod. 2013 Mar;17(1):63-72.

918.97

C₃₉H₅₄N₁₀O₁₄S

23109-05-9

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Room temperature in continental US; may vary elsewhere