Labozol reagent

Total RNA was isolated from each group of hESCs using Labozol Reagent (Cosmo Genetech, Seoul, Republic of Korea) according to the manufacturer’s instructions, and purified RNA was quantified using a NanoDrop spectrophotometer (NanoPhotometer^® N50). cDNA synthesis was performed using 2 μg of total RNA with an M-MuLV reverse transcription kit (Cosmogentech, Seoul, Republic of Korea) and oligo-dT primers. The polymerase chain reaction (PCR) mixture was prepared using EzAmp™ qPCR 2X Master Mix (ELPIS-BIOTECH, Daejeon, Republic of Korea) and run on a Quant Studio™ 3 Real-Time PCR System (Thermo Fisher Scientific). The results were normalized using GAPDH expression as an internal control, and the fold change in gene expression was calculated using the comparative cycle time (Δct) method. The primers used in this study are listed in Table 1.

Huh7 cells were seeded (2 × 10⁵ cells/well) in 12-well plates and treated with various concentrations of pelitinib for 48 h. Total RNA was isolated using Labozol reagent (Cosmogenetech, Seoul, Korea) and 1 μg of RNA was reverse transcribed into complementary DNA (cDNA) using a TOPscript cDNA synthesis kit (Enzynomics, Daejeon, Korea). Quantification of mRNA was performed using a RT-q premix and SYBR Green Q Master (Cosmogenetech, Seoul, Korea) according to the instructions of manufacturer. Quantitative PCR conditions were set as follows: initial denaturation at 95 °C for 5 min followed by between 35–40 cycles of 95 °C for 5 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. GAPDH was used for normalization. Each gene expression level was calculated using the 2^{−∆∆∆Cq} method. The sequences of PCR primers used were listed as follows: TWIST1 sense, 5’-GGC TCA GCT ACG CCT TCT C-3’; TWIST1 antisense, 5’-CTC CTT CTC TGG AAA CAA TGA CAT-3’, GAPDH sense, 5’-GGT GTG AAC CAT GAG AAG TAT GA-3’; GAPDH antisense, 5’-GAG TCC TTC CAC GAT ACC AAA G-3’, CDH1 sense, 5’-TTC CCA ACT CCT CTC CTG-3’; CDH1 antisense, 5’-AAA CCT TGC CTT CTT TGT C-3’, CDH2 sense, 5’-CCT CCA GAG TTT ACT GCC ATG AC-3’; CDH2 antisense, 5’-GTA GGA TCT CCG CCA CTG ATT C-3’.

This study comprised 41 cervical tissue samples. We obtained 26 formalin-fixed paraffin-embedded (FFPE) and 15 frozen cervical tissues samples from the Korea Gynecologic Cancer Bank, Yonsei University College of Medicine, Seoul, Republic of Korea. We divided the collected samples into three control groups (HPV-negative normal tissues [NN], HPV-negative cancer tissue [NC], and HPV16-positive normal tissue [PN]) and one experimental group (HPV16-positive cancer tissue [PC]). Detailed information about these samples is reported in Table 1. All cancer samples were squamous cell carcinomas, which comprise about 80% of all cervical cancers. HPV infection was confirmed using the Abbott RealTime High-Risk HPV PCR assay kit (Abbott Molecular, Abbott Park, IL, USA).
Total RNA was extracted from the frozen tissues using the Labozol reagent (CosmoGenetech, Seoul, South Korea) and from the FFPE tissues using the miRNeasy FFPE kit (Qiagen, Valencia, CA, USA). RNA was quantitated using a Nanodrop2000c spectrometer (Thermo Scientific, Wilmington, DE, USA) and DS-11 (DeNovix, Wilmington, DE, USA) spectrophotometers, and the quantitation and quality of the isolated samples were confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).