Two million cells in 200 μl of Hank’s balanced salt solution were subcutaneously inoculated into the rear flanks of 5-week-old female athymic nude (nu/nu) mice (the Jackson laboratory). Once palpable, tumors were measured using vernier calipers every other day. Tumor volumes were calculated using the formula: length × width × height × 0.5236. When tumor volumes reached about 50 mm3, mice bearing each cell line were sorted into three groups to achieve equal distribution of tumor size in all the treatment groups. Group 1 intraperitoneally received the vehicle [1:1.8:0.4:6.8 mixture of dimethyl sulfoxide (DMSO), Kolliphor RH40, dextrose, and Hepes], whereas groups 2 and 3 received JG-231 (2 and 4 mg/kg body weight per dose, respectively). A375, A375-PLX/R, and Colo-829 received 9, 7, and 12 doses, respectively. At the end of the experiment, animals were euthanized by CO2 asphyxiation. All animal studies were performed according to the protocols approved by the Institutional Animal Care and Use Committee at Medical College of Wisconsin, Milwaukee, WI, USA.

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