Tu 686

The human LSCC TU-686 (Procell Life Science & Technology Co., Ltd.), AMC-HN-8 (BeNa Culture Collection; Beijing Beina Chunglian Biotechnology Research Institute), TU-177 (Jennio Biotech Co., Ltd.) and 293T (Procell Life Science & Technology Co., Ltd.) cell lines were cultured in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (HyClone; Cytiva) and 50 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO₂. Furthermore, the aforementioned cell lines were treated with different concentrations (0, 1, 2.5, 5 and 10 µM) of 5-Aza-2′-deoxycytidine (5-Aza-dC; cat. no. S3196; Selleck Chemicals) for 24, 48 and 72 h at 37°C. Different concentrations of 5-Aza-dC were added to DMEM for treatments.

TU686, TU212, M4e, AMC-HN-8, Hep-2, and LCC laryngeal cancer cells (Procell Life Science & Technology Co., Ltd., Wuhan, China) were cultured in DMEM (Procell Life Science & Technology Co., Ltd.) or RPMI-1640 medium (Hyclone, Merck KGaA). Cancer cells were seeded in 96- well microplates (Corning, Corning, NY, USA) for 24 hours. Then, eupalinilide B was added, and plates were incubated for 48 or 72 hours. MTT solution (Innochem) was added, followed by incubation for 4 hours. OD at 570 nm was measured using a microplate reader (PerkinElmer, Waltham, MA, USA).