Pcw57 rfp p2a mcs

Doxycycline, puromycin, and crystal violet were purchased from Sigma-Aldrich (USA). Human PIERCE1 cDNA was subcloned into the following vectors: pCW57-RFP-P2A-MCS (Addgene, Cambridge, MA, USA) for Doxycycline inducible plasmid, pCS4-3×flag for Flag-tagging, pEGFP-C1 for GFP-tagging. pLNCX-myr-HA-AKT1 plasmid was purchased from Addgene (USA) and used to generate stable cell lines. Lentiviral vectors for shPIERCE1 expression were purchased from Sigma-Aldrich (USA). siRNAs for control, PIERCE1, RICTOR, RAPTOR, and TRIB3 were purchased from GenePharma. Lipofectamine 3000 (Invitrogen, USA), Lipofectamine RNAiMAX (Invitrogen, USA), and jetPEI (Polyplus-transfection SA, France) were used for transfection.

Nek2A (clone ID: 38963) in pJP1563 and NuMA1 (clone ID: 871325) in pLenti6.3/V5-DEST were purchased from DNASU. LentiCRISPR-v2 (52961), pCW57-RFP-P2A-MCS (78933), pCW57-MCS1-P2A-MCS2 (80922), pCDH-EF1-FHC (64874), Flag-TurboID (124646), Tet-pLKO-neo (21916), pcDNA3-Plk4 (41165), pcDNA5-STIL (80266), PGK-H2B-mCherry (21217) and PGK-H2B-eGFP (21210) were purchased from Addgene.
Kinase-dead mutant (K37R) of Nek2A was derived from Nek2A in pJP1563 using Q5 Site-Directed Mutagenesis Kit (NEB) following the instructions of manufacturer. Oligos used for the SDM reaction are given in (Supplementary Table 2). Single guide RNA (sgRNA) oligos (Supplementary Table 3) for CRISPR/Cas9 knockout of Nek2A, C-Nap1 and Rootletin were cloned into LentiCRISPR-v2 as previously described [51 (link)]. Dox-inducible overexpressions of Nek2A and PLK4 were achieved by subcloning to pCW57-RFP-P2A-MCS and pCW57-MCS1-P2A-MCS2 respectively. Nek2A-WT and Nek2A-KD(K37R) cDNAs were subcloned to Flag-TurboID. To perform Co-IP using anti-FLAG antibody, Nek2A cDNA was subcloned into pCDH-EF1-FHC. Oligos for dox-inducible shRNA expression targeting KIF2C were cloned into Tet-pLKO-neo as previously described [52 (link)] and provided in (Supplementary Table 4).