## 3 Results  
To analyze if the expression regulation of Cep135 follows the elimination of centrosomes during oogenesis and their reassembly upon fertilization, we separated _Xenopus laevis_ oocytes according to their size corresponding to different developmental stages (Figure 1A). Ssx2 ip detection by Western blot confirmed increasing expression from immature (stage II) to fully grown (stage VI) oocytes, with a strong increase in fertilizable eggs (Figure 1B,C, Ssx2 ip). Likewise, expression of Cep135 was only seen upon meiotic maturation in mature, nonfertilized eggs (Figure 1B,C, Cep135). This shows that both proteins are expressed in the preparation of fertilization and zygotic centrosome reassembly in maturing oocytes [16]. To investigate if both proteins interact, we immunoprecipitated (IP) Cep135 using specific antibodies directed against an N-terminal peptide of _Xenopus laevis_ Cep135 from native _Xenopus_ egg extracts and identified proteins in immunoprecipitated by tandem mass spectrometry. Mass spectrometry readily revealed Cep135 in IP samples using Cep135 antibodies but not in samples using unspecific IgGs (Figure 1D). Importantly, we also specifically detected Ssx2 ip coprecipitating with Cep135, as well as the previously identified Ssx2 ip interaction partner Wrap73/Wdr8 (Figure 1C). To further evaluate this interaction, we generated antibodies against frog Wdr8. Although the latter did not detect denatured Wdr8 in Western blot, mass spectrometry analysis of its IP fraction specifically showed Wdr8 itself, as well as Ssx2 ip and Cep135 (Figure 1E). The latter IP fractions could also be detected by Western blotting to be specific interaction partners of Cep135 (Figure 1F). Taken together, this suggests that these three proteins build a stable complex in _Xenopus_ egg extracts, most likely to prepare the egg cell for fertilization with the immediate need to rapidly assemble new centrosomes during cleavage divisions in early amphibian development. This is consistent with previous data from _Xenopus_ egg extracts showing an interaction between Ssx2 ip and Wdr8 [6; 13], as well as data from human and mouse cells demonstrating that SsX2 IP interacts with WDR8 and that WDR8, in turn, associates with CEP135 [10; 11; 17]. To better understand if and how SsX2 IP, WDR8, and CEP135 functionally interact with each other in cells with established centrosomes, we generated individual KOs of all three open reading frames in human somatic RPE-1 (retinal pigment epithelial) cells using CRISPR/Cas9 gene targeting. Selected guide RNAs introduced DNA breaks in the third exons of the respective genomic human sequences (Figure 2A, red triangles; please note that 5\({}^{\prime}\)UTRs are also counted as exons). Sequencing of the loci revealed homozygotic loss-of-function mutations for _CEP135_ (Figure S1), _SSX2 IP_ (Figure S2), and _WDR8_ (Figure S3) in CRISPR KO cells (indicated in italics, lower case). Immunoblot analysis using antibodies against CEP135 and SSX2 IP showed reduced signals in several clones of respective KO cells (Figure 2B, please note that WDR8 could not be detected by immunoblot). Consistently, immunofluorescence using specific antibodies against CEP135, WDR8, and SSX2 IP revealed loss of the characteristic centriolar (CEP135) or satellite (SSX2 IP, WDR8) signals in KO cells (Figure 2C; see Figure S4 for analysis of all cell clones). Despite the loss of these gene products, clones of KO cells could be readily identified and grew stably (Figure 2D). While _cep135_ and _wdr8_ KO cells even proliferated like untreated RPE-1 cells, _ssx2 ip_ KO cells showed a mild proliferation delay (Figure 2D, Figure S4; compare _ssx2 ip_ KO with controls and _cep135_ and _wdr8_ KO cells). When we further monitored cell-cycle progression, we found fewer _ssx2 ip_ KO cells in later cell-cycle stages (S/G\({}_{2}\)/M) than in controls and _cep135_ KO cells, while more _wdr8_ KO cells remained in late cell-cycle stages.