Snu 182

SNU-182 and SNU-398 human HCC cell lines (ATCC, USA) were used. FBS was mixed with RPMI 1640 medium with a ratio of 1: 9 to prepare cell culture medium. Cells were cultivated at 37°C. PcDNA 3.1 vectors expressing MACC1-AS1 or TGF-β1, as well as negative control (NC) siRNA and MACC1-AS1 siRNA, were from Sangon (Shanghai, China). Cells were harvested at 70–80% confluence. Cells were counted, and lipofectamine 2000 (Invitrogen, USA) was used to transfect vectors (10 mM, the NC group was empty vector) or siRNAs (45 nM, the NC group was NC siRNA) into 2×10⁶ cells. In all transfections, control (C) cells were untransfected cells. Cells were collected 24 h later for subsequent studies.

The human hepatocyte cell line THLE-2, and HCC cell lines Hep3B, SNU-182, SNU-387, SK-Hep1, and PLC/PRF/5 cells were purchased from ATCC (USA). The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in an incubator at 37°C in 5% CO₂. Cell culture-related reagents were purchased from Gibco (USA). To explore whether CDKN2B could regulate the sensitivity of HCC to Sorafenib, drug intervention was performed on Hep3B and SNU-182 cells. Sorafenib was purchased from Bayer Corporation (West Haven, CT, USA), and dimethylsulfoxide (DMSO) was purchased from KeyGen Biotech Co., Ltd. (Nanjing, China). Sorafenib was dissolved in DMSO and diluted with RPMI 1640 to the desired concentration for in vitro studies. In the control group, DMSO was added to cultures as a vehicle.
The wild-type CDKN2B coding sequence was subcloned into pcDNA3.1 (Sangon Biotech, China) to construct a pcDNA-CDKN2B expression vector. CDKN2B transfections were performed using Lipofectamine 2000 (Invitrogen, USA). siCDKN2B was purchased from GenePharma (China), and the siRNA transfections were performed by Lipofectamine 2000. The transfection efficiencies were detected using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot.