Plasmid Construction and Lentivirus Production

Plasmids expressing wild-type or mutant CycT1 or expressing shCycT1 were described previously (Fu et al., 2022 (link)). NEK7 cDNA was kindly gifted by Prof. Rong Tan (Tan et al., 2017 (link)). The cDNA was amplified by PCR and ligated into the pCDH-CMV-MCS-EF1-Puro vector with EcoRI and BamHI. The shRNA oligos were synthesized (Tsingke Biotechnology) and cloned into the pLKO.1-TRC vector. The shRNA plasmid and packaging vectors (pMD2.G and psPAX2) were transiently co-transfected into 293T cells using Poly(ethyleneimine) solution (Sigma, P3143). The target cells were treated with lentivirus supernatant and 8 μg/ml polybrene for 24 h, followed by 1 μg/ml puromycin (Selleck, s7417) selection for 3 days. On Day 5 after lentivirus infection, cell experiments were performed, and the cell lysates were prepared. All the sequences of shRNA and primers are listed in Supplementary Table S1.