Gentamicin

Recombinant ANKRD22-expressing lentivirus particles (100 µl/ml; multiplicity of infection=100; Cyagen Inc.) were used to infect 293T cells (Cell Bank of Shanghai Branch of the Chinese Academy of Sciences) which were maintained in high-glucose DMEM (Corning, Inc.) supplemented with 10% fetal bovine serum (Corning, Inc.) and 100 µg/ml gentamicin (Sangon Biotech Co., Ltd.) at 37°C in a humidified environment with 5% CO₂. Following incubation for 48 h, cells were passaged and transferred to a new six-well plate and screened with 5 µg/ml puromycin for 7–10 days. After propagation, 293T cells with high expression levels of ANKRD22 were digested with 0.25% trypsin solution (Genom Biotechnology Co. Ltd.) at room temperature for 5 min and collected by centrifugation at 1,000 × g at room temperature for 5 min and washed twice with PBS. Following aspiration of the PBS, 500 µl ice-cold radioimmunoprecipitation assay lysis buffer (pH 7.5; 50 mM Tris-HCl buffer containing 150 mM NaCl, 1 mM EDTA, 0.25% deoxycholate acid and 1% NP-40 and 0.25% deoxycholate sodium) was added, and the solution was mixed by pipetting and gentle agitation. The solution was lysed on ice for 15 min, centrifuged at 13,000 × g at 4°C for 10 min, and the supernatant was aspirated for western blot analysis.

The gastric cancer cells SGC7901 and BGC823 and human THP-1 macrophages were purchased from the Cell Bank of the Shanghai Branch of the Chinese Academy of Sciences. Mouse RAW264.7 macrophages were kindly provided by Professor Hongxiang Sun (College of Animal Sciences, Zhejiang University). Cells were cultured in the following media (Corning, Corning, NY) supplemented with 10% fetal bovine serum (FBS) (Corning) and 100 μg/mL gentamicin (Sangon Biotech, Shanghai, China) at 37°C in a humidified 5% CO₂ atmosphere. Then, SGC7901 and BGC823 cells and human THP-1 macrophages were cultured in RPMI 1640 medium. Meanwhile, the mouse RAW264.7 macrophages were cultured in Dulbecco’s modified Eagle medium (DMEM).