NudCL2 contains a core structure of p23 (p23 domain), which acts as an Hsp90 cochaperone to regulate the folding and maturation of client proteins9. previous work showed that nuclear distribution gene C (NudC)-like protein 2 (NudCL2) localizes to centrosomes; however, little is known about the role of NudCL2 in the regulation of centrosome function. Here, we find that NudCL2 is required for accurate centriole duplication by stabilizing the E3 ligase HECT domain and RCC1-like domain-containing protein 2 (HERC2). Knockout (KO) of using CRISPR/Cas9-based genome editing or Ptgfr depletion of NudCL2 using small interfering RNA causes significant centriole amplification. Overexpression of NudCL2 significantly suppresses hydroxyurea-induced centriole overduplication. Quantitative proteomic analysis reveals that HERC2 is downregulated in KO cells. NudCL2 is shown to interact with and stabilize HERC2. Depletion of HERC2 leads to the similar defects to that in KO and HERC2-depleted cells. Taken together, our data suggest that NudCL2 plays an important role in maintaining the fidelity of centriole duplication by stabilizing HERC2 to control USP33 protein levels, providing a previously undescribed mechanism restraining centriole amplification. in mammalian cells. A CRISPR/Cas9 plasmid with a short guide RNA (sgRNA) that recognizes the first Gemigliptin exon of was constructed and transfected into U2OS cells (Fig. ?(Fig.2a).2a). PCR amplification of genomic DNA followed by Sanger sequencing revealed indels that are predicted to cause frameshift mutations at the DNA locus (Fig. ?(Fig.2b).2b). Immunoblotting confirmed that NudCL2 protein disappeared in the mutant cells (Fig. ?(Fig.2c).2c). In knockout (KO) cells at interphase, the number of cells with more than four centrin, four CP110, or two -tubulin dots increased approximately three-fold compared to the wild-type (WT) cells (Fig. 2dCh), suggesting that loss of NudCL2 causes centriole amplification. The similar results were observed in KO DLD1 Gemigliptin cells and NudCL2-depleted CAL51 cells (Supplementary Figs. 1 and 2). Moreover, the increase in centriole number observed Gemigliptin in KO cells was significantly reversed by ectopic expression of NudCL2 (Fig. 2iCl). Given that cell cycle arrest may induce centriole amplification2,11, we determined whether centriole amplification induced by NudCL2 deletion resulted from a change in cell cycle progression in KO cells. Fluorescence-activated cell sorting (FACS) analysis showed that there was no significant difference between the WT and KO cells (Fig. 2m, n). Together, these data indicate that NudCL2 plays an important role in restraining centriole amplification. Open in a separate window Fig. 2 Downregulation of nuclear distribution gene C-like protein 2 (NudCL2) leads to centriole amplification.a Schematic representation of gene targeting strategy. b Indel mutations of the DNA locus in two knockout cell lines. c Western blot analysis of NudCL2 protein in control and KO U2OS cells. -actin, a loading control. dCf Control and KO U2OS cells were fixed and processed for immunofluorescence analysis with anti-centrin (green) and anti-CP110 (red) antibodies. Higher magnifications of the Gemigliptin boxed regions are displayed. The frequencies of cells with more than four centrin and four CP110 dots were calculated, respectively. g, h Control and KO U2OS cells were fixed and stained with anti–tubulin (green) and anti-CP110 (red) antibodies. Higher magnifications of the boxed regions are shown. The number of cells with more than two -tubulin dots was plotted. iCl Control and KO U2OS cells were transfected with green-fluorescent protein (GFP)-NudCL2 or GFP vector for 48?h and subjected to western blot and immunofluorescence analyses, respectively. -actin, a loading control. The frequencies of cells with more than four centrin, four CP110, and two -tubulin dots were plotted, respectively. m, n The cell cycle distribution of control and KO U2OS cells was analyzed by flow cytometry. o, p Cells were fixed and immunostained with anti–tubulin (green) and anti-CP110 (red) antibodies. Representative images of mitotic cells with bipolar, pseudobipolar, multipolar, or monopolar spindles are shown. The percentages of cells with various mitotic phenotypes were calculated. DNA was visualized with Gemigliptin 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 10?m. Quantitative data are expressed as the mean??SD (at least three independent experiments). More than 150 cells were counted in each experiment. *test Centrosomes are essential for bipolar spindle assembly and accurate chromosome segregation in mammalian cells1. Centriole amplification leads to supernumerary centrosomes in the subsequent cell cycle, which cluster to generate pseudobipolar spindles after transient spindle multipolarity, promoting chromosome missegregation2,12C15. To investigate the effects of NudCL2 deletion on mitotic spindle formation, we performed immunostaining analysis with anti–tubulin and anti-CP110 antibodies. The data showed that the frequency of cells exhibiting pseudobipolar spindles was significantly increased in KO cells compared with that in WT cells (Fig. 2o, p), implying that loss of NudCL2 influences the formation of bipolar spindles. Overexpression of NudCL2 suppresses centriole overduplication Treatment with hydroxyurea (HU, a DNA synthesis inhibitor) uncouples centriole duplication from DNA replication and induces multiple rounds of centriole duplication in a prolonged S phase.