For cisplatin or Salinomycin IC₅₀ analysis in SW620 cells or Cisp-resistant SW620 cells, cells (1×10⁴/well) are cultured in 96-well plates and treated with different chemotherapeutics (cisplatin, Salinomycin) in different concentrations for 48 h. Then 20 μL of cell counting kit-8 (CCK-8) is added into each of the 96-wells. After 4 h incubation at 37°C, the optical density (OD) values are detected at 450 nm using the scan reader. Cell growth inhibiting rates are described as cell inhibiting curves and the IC₅₀ parameters (inhibiting concentration of 50% cells) are evaluated by Xlfit 5.2 software. For cell proliferation analysis, SW620 cells or Cisp-resistant SW620 cells (5×10³/well) are also seeded in 96-well plates in serum-containing medium and treated with cisplatin (5 μM, according to the calculated IC50 values of cisplatin in SW620 cells) for 0, 12, 24, 48, 72 and 96 h. Then 20 μL cell counting kit-8 is added into each of the 96-wells. After 4-h incubation at 37°C, the coloring reactions are also quantified at 450 nm^[2].

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

Mice^[3]
Nude mice (nu/nu; 4-6 weeks of age) are used. HepG2 cells are suspended in 100 mL 1:1 serum-free DMEM and Matrigel. Mice are anesthetized with ketamine/xylazine and after surgically opening the abdomen, HepG2 cells are inoculated into the liver parenchyma and mice are monitored every 3 days for 35 days. Finally, 18 nude mice are divided into three groups that are intraperitoneally injected daily for 6 weeks: two Salinomycin-treated groups (4 mg/kg Salinomycin group, 8 mg/kg Salinomycin group) and the control group (saline water group).
Rats^[4]
A total of 10 male rats are used in the experiment. After a routine anesthesia, the abdomen is opened. After a resuspension of high glucose medium not containing serum DMEM, and matrigel, the bladder transitional cancer cell line T24 is inoculated in the parenchyma of bladder in rats, and then the abdomen is sutured. After operation, the rats are randomized into the experiment group and the control group with five in each group. After operation, the rats in the experiment group are immediately given intraperitoneal injection of Salinomycin with a dosage of 8 mg/kg, while the rats in the control group are given intraperitoneal injection of normal saline. A close observation is paid during the drug administration period. After 15 d, the rats are sacrificed by cervical dislocation, and the complete tumor tissues are stripped to observe the tumor growth and metastasis.

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

• [1]. Lu D, et al. Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells. Proc Natl Acad Sci U S A. 2011 Aug 9;108(32):13253-7.

  [2]. Zhou J, et al. Salinomycin induces apoptosis in cisplatin-resistant colorectal cancer cells by accumulation of reactiveoxygen species. Toxicol Lett. 2013 Oct 24;222(2):139-45.

  [3]. Wang F, et al. Salinomycin Inhibits Proliferation and Induces Apoptosis of Human Hepatocellular Carcinoma Cells In Vitro and In Vivo. PLoS One. 2012; 7(12): e50638.

  [4]. Qu H, et al. Effect of salinomycin on metastasis and invasion of bladder cancer cell line T24. Asian Pac J Trop Med. 2015 Jul;8(7):578-82.

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