Quantitative real‑time polymerase chain reaction
KYSE150 cells were treated with DMSO (0.1%) or ixazomib (10 μM) for 24h. Total RNA was isolated using the Trizol reagent and Ultrapure RNA kit (CW Biotech, China) according to the manufacturer’s instructions. 2 mg of total mRNA was reverse-transcribed with the Superscript™ reverse transcription system (Takara, Dalian, Japan). Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) reaction was carried out on ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR master mix reagents (Takara, Dalian, Japan). Relative quantification of target gene mRNA level was calculated after normalization to GAPDH mRNA using the 2-△△Ct method. The primer sequences were selected based on the published literature (Du et al. , 2017).