Tu177

Three laryngeal cancer cell lines (TU177, TU686 and TU212) were purchased from American Type Culture Collection. All cell lines were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.), while AMC-HN-8 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) under conventional culture conditions. All cell lines were authenticated via DNA fingerprinting using the short tandem repeat (STR) method from Procell Life Science & Technology Co., Ltd. The cDNA of 10 LSCC-adjacent tissues were randomly mixed in equal proportion as the control group (pools). For RNA sequencing, TU177 cells were starved in serum-free medium overnight before the addition of recombinant TGF-β1 (10 ng/ml, R&D Systems, Inc.), and cells were routinely cultured for 7 days at 37°C in a humidified atmosphere with 5% CO₂. Total RNA from TU177 cells was used for sequencing on a HiSeq 2000 system (Illumina, Inc.). Cuffdiff (version 2.2.1.2) was used to compare the log ratio of Fragments Per Kilobase of transcription per Million mapped reads in the two conditions (11 (link)). The differential expression results were constructed by GraphPad Prism7 software (GraphPad Software, Inc.).

Three human LSCC cell lines (TU686, TU177 and AMC-HN-8) and 293T cells were purchased from BNBIO (Beijing, China) and preserved at the Otorhinolaryngology Head and Neck Surgery Biobank of Hebei Medical University. The TU686 and TU177 cells were cultured in RPMI-1640 medium (Gibco/Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Gibco/Thermo Fisher Scientific, Inc.). The AMC-HN-8 and 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco/Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. The TU177 cells were treated with 10 ng/ml recombinant TGF-β (R&D Systems, Inc., Minneapolis, MN, USA) for 7 days and the medium was replenished every 2 days. All the cells were cultured at 37°C in a humidified 5% CO₂ incubator (Thermo Fisher Scientific, Inc.).