## 3 Results
Taken together, this shows that none of these gene products is individually essential for survival in non transformed human RPE-1 cells, and that their complete loss of function leads to only marginal cell proliferation defects. Figure 1: Interactome of Cep135, Wdr8, and Ssx2 ip in _Xenopus_ egg extracts. (**A**) Schematic drawing illustrating the state of centrosomes in somatic cells, developing oocytes, ovum (egg), and fertilized egg (zygote). (**B**,**C**) Immunoblotting (**B**) and corresponding quantification (**C**) of Ssx2 ip, Cep135, and \(\alpha\)-tubulin to evaluate protein expression at different _Xenopus_ oocyte stages. The total amount of protein was controlled by glycerol aldehyde 3 phosphate dehydrogenase (Gapdh). (**D**,**E**) Identification of proteins specifically precipitated with antibodies against Cep135 (**D**) or Wdr8 (**E**) using tandem mass spectrometry. The list shows only a fraction of proteins identified. (**F**) Immunoprecipitations using antibodies against Wdr8 (elu1 Wdr8, elu1 IgG: control) analyzed by immunoblotting of Cep135 and Ssx2 ip. Input (total extract) and supernatants (sup1/sup2) of extracts after IP are shown from two sequential precipitations. To evaluate the interdependent centrosomal localization of all three proteins, we next monitored and compared their expression and subcellular distribution in wildtype and individual KO cells. Although expression of CEP135 was reduced in _ssx2 ip_ and _vdr8_ KO cells (Figure 3A), its centrosomal localization remained unaffected (Figure 3B,C). In contrast, SSXZIP-containing centriolar satellites were seen at a significantly larger distance from centrosomes in _vdr8_ and _cep135_ KO cells (Figure 3D,E). Consistently, a more dispersed distribution of the centriolar satellite marker PCM1 was observed in _ssx2 ip_, _vdr8_, and _cep135_ KO cells (Figure S5). Of note, satellites were even more dispersed in frequently observed _cep135_ KO cells with two separated centrosomes (Figure 3D,E, compare _cep135_ KO with _cep135_ KO split pair). This shows that satellites defined by SSXZIP or PCM1 require centrosomal localization of CEP135 to efficiently localize around centrosomes. In contrast,  
Figure 2: _cep135_, _vdr8_, and _sex2 ip_ KOs generated by CRISPR/Cas9 in human RPE-1 cells line are vital and proliferate. **(A)** Genomic organization of human _CEP135_, _WDR8_, or _SSXZIP_ loci with indicated targeting sites used for CRISPR/Cas9-mediated DNA cleavage. **(B)** Immunoblot to detect CEP135 (upper panel) or SSXZIP (lower panel) in cell clones after KO of the respective genes. Please note that the SSXZIP antibody tended to reveal unspecific signals in some immunoblots (see Figure 3A). Clones _vdr8_ ko308, _ssx2 ip_ ko2-13, and _cep138_ ko8 were used for all further experiments. **(C)** Immunofluorescence to detect CEP135, WDR8, or SSXZIP in human RPE-1 wildtype (WT) and representative _cep135_, _vdr8_, or _ssx2 ip_ KO cell lines (see Figures S1–S3 for all cell clones). **(D)** Cell proliferation in WT and _vdr8_, _ssx2 ip_, and _cep135_ RPE-1 KO cell lines. A total of 10\({}^{5}\) cells (3.5 cm cm\({}^{2}\) dish) were seeded and counted at indicated times (20 h, 32 h, 44 h, and 56 h). **(E,F)** DNA content analysis using propidium iodide (PI) staining to analyze cell-cycle progression in WT and _vdr8_, _ssx2 ip_, or _cep135_ RPE-1 KO cell lines. The indicated range shows cells in S/G\({}_{2}\)/M-phase. **(F)** Quantification of the fraction of cells in S/G\({}_{2}\)/M-phase. The graph shows average values from independent experiments \(\pm\) S.D. ; the _p_-value was calculated using a two-tailed Student’s _t_-test. * indicates \(p\) < 0.05, *** \(p\) < 0.001. CEP135 localizes independently of SSX2 IP and WDR8 to centrosomes, while its expression still relies on these two proteins.