## 3 Results
To further investigate the frequently observed centrosome splitting in _cep135_ KO cells (Figure 4A, arrowheads), we monitored and quantified cells with split centrosomes using \(\gamma\)-tubulin signals separated by more than 1 \(\upmu\)m (Figure 4B) in WT and individual _cep135_ KO cells at different cell-cycle stages. Of note, splitting occurred independently of the cell-cycle stage. Cells that were withdrawn from the cell cycle upon serum starvation and, thus, remained in G\({}_{0}/_{1}\) phase showed a similarly high number of split centrosomes as unsynchronized cells or cells that were blocked in S-phase using high thymidine concentration (Figure 4B). In contrast, split centrosomes remained rare in WT cells, as well as in _sxx2 ip_ and _wdr8_ KO cells (Figure 4B). While the fraction of cells showing premature split centrosomes was lowest in control cells in G\({}_{0}/_{1}\) (1.7%), premature centrosome splitting increased to  
Figure 3: Protein expression and centrosomal localization of CEP135, WDR8, and SSX2 IP are interdependent in RPE-1 cell lines. (**A**) Immunoblotting to show expression levels of PCMI, CEP135, SSX2 IP, and \(\gamma\)-tubulin. \(\alpha\)-Tubulin was used as a control for total protein loaded. Please note that the SSX2 IP antibody tended to reveal unspecific signals in some immunoblots. (**B–E**) Centrosomal CEP135 (**B,C**) and SSX2 IP (**D,E**) signals in WT and KO cells as indicated. _cep135_ KO cells frequently displayed separated centrosomes with two distinct \(\gamma\)-tubulin signals (split pair). Representative immunofluorescence images (**C,E**, both with \(\gamma\)-tubulin signals) and quantifications (**B,D**). The ImageJ threshold feature was used to identify the region of the CEP135 signal in (**B**). A square (8.6 \(\upmu\)m in _xy_) was drawn with the \(\gamma\)-tubulin signal in the center in (**D**). (**B,D**) Graphs show mean values (fat colored/black dots) of single experiments and individual data points (transparent dots). The added intensity of the two separated \(\gamma\)-tubulin signals (“split pair”) in _cep135_ KO cells was quantified in (**D**). Three or four independent experiments were performed with more than 500 (**B**) or 210 (**D**) cells measured in each experiment. Errors bars show the SD from means; _p_-values were calculated with a two-tailed Student’s \(t\)-test. * indicates \(p<0.05\), ****\(p<0.001\). The surface plots represent sums (Image) _z_-projections) of all images. (**C,E**) Scale bars: 5 \(\upmu\)m (**C**) or 2 \(\upmu\)m (**E**). more than 43% in _cep135_ KO cells and, after nocodazole treatment to depolymerize MT, even up to 68% (7% in WT controls Figure 4C). At the same time, nocodazole treatment increased the intercentrosomal distance from an average of 2.0 \(\upmu\)m to 4.3 \(\upmu\)m.  
Figure 4: _cep135_ KO cells show premature centrosome splitting and diminished PCM. (**A**) \(\upgamma\)-Tubulin was stained to identify cells with premature split centrosomes and reduced PCM in WT and _cep135_ KO cells. Scale bar: 20 \(\upmu\)m. (**B**) Quantification of centrosome splitting by more than 1 \(\upmu\)m at different cell-cycle stages in _cep135_, _wdr8_, and _ssex2 ip_ KO cell lines. (**C**) Quantification of centrosome distance in WT and _cep135_ KO cells at G\({}_{1}\) (48 h starvation) before and after nocodazole treatment that greatly increased centrosome distances in _cep135_ KO cells. (**D**,**) Quantification of the centrosomal \(\upgamma\)-tubulin (**D**) and CDKSPAP2 (**E**) signals in WT, _wdr8_, _ssex2 ip_, and _cep135_ KO cells. _cep135_ KO cells displaying two distinct \(\upgamma\)-tubulin signals were quantified separately either using summed intensities of both centrosomes (split-pair) or as separated values for each of the two individual centrosomes (split single). (**D**) Cells in G\({}_{0/1}\) and S-phase. (**E**) G\({}_{0/1}\) cells. (**B**–**E**) Graphs show mean values (fat colored/black dots) of single experiments and individual data points (transparent dots). Around 300 cells were measured for each cell line.