Pcdh cmv mcs ef1 gfp puro vector

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.

NUSAP1-specific short hairpin RNAs (shRNAs) were purchased from Sigma-Aldrich and then cloned into the PLKO.1 vector. The sequences are as follows: shNUSAP1#1: 5′-CCGGCCTCAGGTAACAGAGATTCAACTCGAGTTGAATCTCTGTTACCTGAGGTTTTTTG-3′; shNUSAP1#2: 5′-CCGGGAGCACCAAGAAGCTGAGAATCTCGAGATTCTCAGCTTCT TGGTGCTCTTTTTTG-3′; and shNUSAP1#4: 5′-CCGGGAACCACACAAAGGAAAGCTACTCGAGTAGCTTTCCTTTGTGTGGTTCTTTTTTG-3′. The sequences encoding human NUSAP1 included the full length of NUSAP1 (amino acids 1–441), the N-terminus of NUSAP1 (amino acids 1–158), the C-terminus of NUSAP1 (amino acids 159–441), and NUSAP1-ΔSAP (amino acids 41–441), which were cloned into the pCDH-CMV-MCS-EF1-GFP-Puro vector and were provided by GeneCreate. For transient transfection, plasmids encoding full-length ATR and truncated NUSAP1 were collectively transfected into 293FT cells by Lipofectamine 2000. Stable transfection was performed as described previously.^{43 (link)}