Culturesure

A palmitic acid stock solution was prepared using a previously described method [74 (link)]. Briefly, a 100 mM solution of PA (P0500; Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M NaOH solution (194-02191; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was heated at 70 °C in a shaking water bath. In an adjacent water bath, at 55 °C, a 10% (w/v) PA-free BSA solution (CultureSure, 034-25462; FUJIFILM Wako Pure Chemical Corporation) was prepared in ddH₂O. A 5 mM PA/10% (w/v) BSA stock solution was prepared by adding 250 μL of the 100 mM palmitate solution dropwise to 4.75 mL of the 10% (w/v) BSA solution at 55 °C, followed by vortex mixing for 10 s and 10 min incubation at 55 °C. The PA/BSA complex solution was cooled to room temperature and sterile filtered (0.45 μm pore size membrane filter). At the same time, the PA-free BSA stock solution was prepared as a control. The complex solution was stored at −20 °C, where it was stable for 3–4 weeks. The stored 5 mM PA/10% BSA stock solutions were heated for 15 min at 55 °C and then cooled to room temperature before use.

In this study, we used cells from the following two COM cell lines: KMEC (primary-tumor site of origin; oral gingiva) and LMEC (metastatic site of origin; lymph node) cell lines. One co-author, Dr. Takayuki Nakagawa of Tokyo University, provided the cells. Cell culture methods and protocols accorded with the published note on these cell lines [34 (link)]. Briefly, cells were cultured using Roswell Park Memorial Institute (RPMI) media-1640 (Gibco), l-glutamine solution (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), antibiotics (penicillin-streptomycin; Sigma), and 10 % fetal bovine serum (BI, Biological Industries), and maintained at 37 °C in a controlled-humidity environment with 5 % CO₂. The cells were then stored in liquid nitrogen with a media supplement (CultureSure®, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). Cold phosphate-buffered saline (PBS) and 0.25 % trypsin or 0.1 % EDTA were applied during the subculture. Cells were counted using an automated cell counter (LUNA II, Logos, USA).

COM tissue specimens were acquired from tumours excised from dogs that had undergone surgery at the Veterinary Teaching Hospital of Kagoshima University. Informed consents were obtained from dog owners. COM patient’s information is presented in Table S1a. Normal oral tissues were collected from healthy laboratory beagle dogs (age range 8–10 years) at Kagoshima University. Experimental conditions and design were approved by Kagoshima University and Veterinary Teaching Hospital ethics committee (KV004; 18.04.2011). All experimental methods were carried out in accordance with the approved guidelines and regulation.
Tissue samples were collected immediately after excision from dogs that had undergone surgery. The diagnosis was confirmed histopathologically by the hospital. The tissue specimens were placed in RNAlater (AM7021, Invitrogen, Carlsbad, CA, USA) immediately after isolation and stored at −80 °C after overnight incubation at 4 °C.
Dog melanoma cell lines KMEC and LMEC were stored in freezing medium (039-23511, CultureSure, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). Cell lines were cultured according to the procedure described previously [21 (link)]. Cells were grown until confluence and then RNA was extracted for evaluation.