KIFC1 Knockout and Overexpression in 293T Cells

The human 293T cells (ATCC, CRL-3216) and two kifc1 knockout cell lines were cultured in a 37 °C incubator with 5% CO₂ in standard cell culture medium (Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin) (all from GIBCO, USA). The full length KIFC1 was cloned and inserted with BamH I restriction site into a commercial expression vector (pCMV-N-Flag; Beyotime, D2722), and the plasmid DNA was transfected in cells at approximately 70% confluence using Lipo6000 reagent (Beyotime, C0526). According to the manufacturer’s instruction, after incubated the overexpressed/rescued cells for 36 h, we then harvested the cells for western blot and cell cycle analysis. For inhibitor treatment, the small molecule compounds CW069 (100 µM; Cayman, USA) and AZ82 (0.5 µM; Cayman, USA) were supplemented in cultural medium as previously described^{34 (link),35 (link)}.