Sune1

Human nasopharyngeal epithelial cell line NP69 was acquired from Sigma-Aldrich (SCC197, St. Louis, MO, USA) and grown in keratinocyte serum-free media (17,005,042, Gibco, Rockville, MD, USA) with 10% fetal bovine serum (FBS, 10,082,147, Gibco) and 1% penicillin-streptomycin (P/S) solution (PB180120, Procell) in an incubator with 5% carbon dioxide (CO₂) at 37 °C. NPC cell lines from American Type Culture Collection (ATCC, Manassas, VA, USA), such as C666-1 (ACS-5006) and SUNE-1 (CRL-5971) were purchased and grown in RPMI-1640 (30-2001, ATCC) with 10% FBS and 1% P/S at 37 °C with 5% CO₂.

CNE-1, HONE-1, SUNE-1, 5-8F cells, human nasopharyngeal epithelial NP69 cells were all purchased from ATCC (Manassas, VA, USA). The culture condition of the cells was RPMI-1640 medium plus 10% fetal bovine serum (FBS, Invitrogen, Shanghai, China) and 1% streptomycin/penicillin (Hyclone, South Logan, UT, USA) at 37 °C with 5% CO₂. NP69 cells were maintained in keratinocyte/serum-free medium (Invitrogen, Shanghai, China) with bovine pituitary extract (Absin Bioscience Co., Ltd., Shanghai, China). All cells were incubated in a humidified CO₂ (5%) incubator at 37 °C.

Four NPC cell lines (SUNE1, CNE2, HNE1 and C666) and an immortalized nasopharyngeal epithelial cell line (NP69) were ordered from American Type Culture Collection (ATCC). All cell lines were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, penicillin (100 U ml⁻¹) and streptomycin (100 U ml⁻¹), and maintained in a humidified 5% CO₂ incubator at 37°C. The cisplatin-resistant NPC cells (HNE1/DDP and CNE2/DDP) were established as previously reported [12 (link)]. Briefly, parental cell lines CNE1 and CNE2 were cultured with gradually increasing concentrations of cisplatin for six months. The initial concentration of cisplatin was 0.02 µg ml⁻¹ and the final concentration was 1 µg ml⁻¹. Cisplatin resistance was confirmed by the in vitro toxicity test.

Human nasopharyngeal carcinoma cell line Sune-1 was purchased from ATCC(American type culture collection). Sune-1 cells were cultured in DMEM medium (ThermoFisher, USA) containing 10% fetal bovine serum (ThermoFisher, USA), 100 U/mL penicillin and 100 U/mL streptomycin. Cells were maintained in a humid atmosphere with 5% CO₂ at 37 °C.

Human nasal epithelial cells (HNECs) and NPC cells (SUNE1, CNE2, HK1, and HONE1) purchased from the American Type Culture Collection (ATCC) were cultured in 5% CO₂ animal cell incubator at 37°C under a culture medium system containing Dulbecco’s modified Eagle’s medium (DMEM) (HyClone Company), 10% fetal bovine serum solution (Gibco Company), and 1% penicillin–streptomycin solution (100×, Solarbio Company). Subsequent experiments were carried out after the cells were cultured to reach 80–90% confluence.
Before transfection, the culture medium was replaced with a culture medium without fetal bovine serum, and on the day of transfection, the cells were seeded into a six-well plate at 1 × 10⁵ cells/well. The miR-34c-5p mimics, miR-34c-5p inhibitor, NC mimics, NC inhibitor, NOTCH1 siRNA, and NC siRNA vectors were all purchased from Shanghai Sangon Biotech Co., Ltd. The cell lines were transfected with a Lipofectamine 2000 transfection kit (Invitrogen, United States) in strict accordance with the kit instructions. After 8 h of transfection, the culture medium was replaced with fresh culture medium at 37°C/5%CO₂.