Lentiviral Knockdown and Overexpression Constructs

The negative control plasmid pLKO.1-shGFP was purchased from Addgene (Massachusetts, USA). The shNUCKS sequences (Table S1) were inserted into the pLKO.1 vector. As described previously, the cDNA sequence of human NUCKS was purchased from Youbio Biological Technology Co., Ltd. (Changsha, China), which was cloned into the pCDH-CMV-MCS-EF1-GFP-Puro vector provided by GeneCreate (Wuhan, China) [27 ]. The Beclin1 shRNA plasmid was purchased from Sigma-Aldrich (TRCN0000299864), and the plasmid GFP-LC3B [28 (link)] was a gift from Prof. Ning Gao at the Third Military Medical University, China. Lentiviruses were generated by co-transfecting the 293FT cell line with the packaging plasmids pLP1, pLP2, and pLP/VSVG and the corresponding shRNA plasmids. Lipofectamine 2000 was used for all transfections according to the manufacturer’s instructions. Virus-containing supernatants were harvested and used for cell infections at a final concentration of 4 mg/ mL polybrene. At 24 h after the final round of infection, the cells were cultured in the presence of 2 mg/mL puromycin, and the drug-resistant cells were selected and pooled for subsequent experiments.