Ov hoxb2

HFWT cells were cultured in a 6-well plate at a density of 1×10⁶ cells/well at 37°C in a 5% CO₂ incubator for 24 h. The lentiviral vectors packaging short hairpin RNA (shRNA) NUSAP1 (sh-NUSAP1), negative control shRNA (sh-NC), HOXB2 overexpression plasmid (Ov-HOXB2) and Ov-NC were obtained from Shanghai GeneChem Co., Ltd., and were then transduced into cells at 20 nM using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The transfection efficiency was verified by RT-qPCR and western blot analyses after 48 h.

Normal intestinal epithelial cells (HIEC cells) and colon cancer cell lines (Caco-2, LoVo, HCT116 and SW480 cells) were obtained from the American Type Culture Collection. All cell lines were cultured in RPMI-1640 medium (HyClone; Cytiva) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). Cells were maintained in a humidified incubator at 37°C with 5% CO₂. Cells were transfected with 20 nM CCT6A knockdown [short hairpin (sh)RNA-CCT6A#1 and shRNA-CCT6A#2] and interference control (shRNA-NC), HOXB2 overexpression (Ov-HOXB2) and overexpressed control group (Ov-NC), which were purchased from Shanghai GeneChem, Co., Ltd. Polybrene (Shanghai GeneChem, Co., Ltd.) was used as a transfection reagent. Transfections were performed using Lipofectamine^® 2000 according to the manufacturer's protocol. After transfection for 48 h at 37°C with 5% CO₂, transfection efficiencies were assessed via reverse transcription-quantitative PCR (RT-qPCR) and western blotting after 48 h.