Mycoplasma pcr detection kit

HepG2 cells obtained from Korean Cell Line Bank (KCLB) were grown in DMEM medium (Welgene, LM-001-05)-1% penicillin–streptomycin–amphotericin B mixture (Lonza, 17-745E) supplemented with 10% fetal bovine serum (Corning, 35-010-CV). Cells were tested for mycoplasma contamination using a Mycoplasma PCR Detection Kit (e-MycoTM, 25236, iNtRON Biotechnology). PA stock solution was prepared by dissolving palmitate in 70% ethanol and heating at 56 °C. PA stock solution was diluted in 2% fatty acid-free BSA-DMEM before treatment. For in vitro treatment, following concentrations were employed: OGBD, 100 μg/ml; Fluo-3 AM, 5 μM; MitoSOX^TM Red, 5 μM; SYTOX Green Nucleic Acid Stain, 1 μM; BAPTA-AM, 30 μM; calcein-AM, 1 μM; palmitic acid (PA), 500 μM; decylubiquinone (DUB), 200 μM; cobalt chloride (CoCl₂), 2 mM; Gly-Phe β-naphthylamide (GPN), 200 μM; BTP2, 10 μM; MitoTEMPO, 10 μM; EGTA, 2 mM; CA-074Me, 10 μM.

PK-8, PK-45P, T3M-4, and KP4 human PDAC cells were provided by the RIKEN BioResource Research Center through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. PK-59, PK-1, PANC-1, and MIA PaCa-2 human PDAC cell lines were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). The characteristics of the eight PDAC cells are summarized in the Supplementary Table S1^{9 (link),26 (link)–34 (link)}. HPDE6 cells were kindly provided by Prof. Toru Furukawa (Tohoku University, Sendai). Cells were grown in growth medium (RPMI-1640 medium containing 10% fetal bovine serum) at 37 °C in a humidified 5% CO₂ atmosphere. To form spheres, cells (3 × 10³ cells/well) were plated in 96-well ultra-low attachment plates (Thermo Fisher Scientific, Waltham, MA, USA) with growth medium. After 7 days, the spheres were photographed using a phase contrast microscope (Eclipse TS-100, Nikon, Tokyo, Japan). Spheres were then aspirated using micropipettes and used for further experiments. Using a Mycoplasma PCR Detection Kit (iNtRON Biotechnology Inc., Jungwon-Gu, South Korea), it was confirmed that all cells were not infected with mycoplasma.

PTC cell line (SNU790) was purchased from Korean Cell Line Bank (KCLB, Seoul, Korea). Anaplastic thyroid carcinoma cell line (HTH83: established by Dr Nils-Erik Heldin, University of Uppsala, Sweden) was kindly provided by Dr Yoon Woo Koh (Department of Otorhinolaryngology, Yonsei University, Korea, cells authentication was performed by short tandem repeat (STR) analysis) and maintained in complete Roswell Park Memorial Institute medium (RPMI) media with 10% fetal bovine serum (FBS; Gibco BRL). Mycoplasma contamination of the cell lines used for this study was examined with Mycoplasma-PCR Detection Kit according to manufacture's instructions (#25235, iNtRON Biotechnology Inc., Seongnam, Korea).