Female NOD.Cg-Prkdcscid Il2rgtm1wjl/SzJ mice [commonly known as nonobese diabetic (NOD)/SCID gamma mice; The Jackson Laboratory, Maine], 7 to 8 weeks of age and weighing 20 to 22 g, were used in the study of Mel270 cells, whereas female athymic nude mice [NU(NCr)-Foxn1nu mice] (Charles River), 4 weeks of age and weighing 20 to 22 g, were used in the study of A375 cells. All mice were housed in appropriate sterile filter-capped cages and provided with food and water ad libitum. All procedures were essentially performed as previously described (26, 30). Briefly, exponentially growing cultures were harvested, washed, and resuspended in RPMI 1640, and 2 × 106 viable cells were transplanted subcutaneously into the flanks of the mice. For tumor growth analysis, tumor volume was assessed as (LW2/2), where L and W represent the length and the width of the tumor. The animals were monitored twice weekly for tumor development. Results of animal experiments are expressed as means ± SEM of a total of five tumors for Mel270 cells and six tumors for A375 cells. To administer FR to mice, the chemical was first dissolved in dimethyl sulfoxide (DMSO) to make a stock solution (2 mg/ml). Water was then used as the solvent to prepare the injection suspension (100 μg/ml). The final mixture was applied to the treatment group mice (100 μl per mouse, intraperitoneal injection), and vehicle was applied to the control group mice. This xenograft study was approved by the Animal Care and Use Committee, National Institute of Dental and Craniofacial Research and was in compliance with the Guide for the Care and Use of Laboratory Animals.

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