## 3 Results
While the number of abnormally arranged MT arrays was negligible in control and rescue RPE-1 cells, _cep135_ KO cells frequently displayed abnormal MT organization (Figure 7B, please note that the frequency of all abnormal MTOCs (35%) matched the frequency of prematurely spilt centrosomes). In particular, dispersed MT organization (type II) was consistent with PCM decrease and instability. Considering the frequently forming asymmetric structures in MT regrowth assays, and the \(\gamma\)-tubulin and CDK5 RAP2's asymmetric distribution in _cep135_ KO cells (Figure 4F), we further evaluated the imbalance of centrosomal proteins _cep135_ KO cells with split centrosomes. The two distinct \(\gamma\)-tubulin and CDK5 RAP2 signals in interphase cells with separated centrosomes were quantified, and their ratios (higher/lower value) were determined (Figure 7C,D). Indeed, the distribution of ratios in _cep135_ KO compared to WT cells revealed a strong asymmetry in CDK5 RAP2 signals and, to some extent, \(\gamma\)-tubulin signals (Figure 7C,D). Figure 5: C-NAP1 and rootletin signals in prematurely split centrosomes of _cep135_ KO cells. (**A**) Representative immunofluorescence images showing the signal of centrosome linker proteins C-NAP1 and rootletin in WT and _cep135_ KO cells. _cep135_ KO cells frequently displayed split centrosomes. Scale bar: 10 \(\upmu\)m or 2 \(\upmu\)m (insets). (**B**) Quantification of C-NAP1 and rootletin signal intensities in WT and _cep135_ KO cells. Graphs show mean values (fat colored/black dots) of single experiments and individual data points (transparent dots) with error bars representing the SD from three independent experiments. _cep135_ KO cells displaying two distinct \(\gamma\)-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). A total of 70 cells were quantified in each experiment. The \(p\)-value was calculated with a two-tailed Student’s \(t\)-test. ** indicates \(p<0.01\), ***\(p<0.005\), ***\(p<0.001\). (**C**) Immunobloting showing protein expression of NEK2, C-NAP1, and rootletin. \(\alpha\)-Tubulin was used as a loading control. Given the decrease in and disorganization of PCM, as well as the high frequency of readily visible, abnormal MT growth arrays in _cep135_ KO cells, we hypothesized frequent mitotic defects, although such a hypothesis was inconsistent with the normally proliferating _cep135_ KO cells showing regular cell-cycle progression (Figure 2D-F). However, the analysis of mitotic spindle poles showed an apparently equal distribution of both \(\gamma\)-tubulin and CDK5 RAP2 in individual cells (Figure 8A). In fact, the overall population of cells in mitosis showed an increased (although not significantly) accumulation of \(\gamma\)-tubulin and CDK5 RAP2 (Figure 8B,C), in contrast to what we observed in interphase (Figure 7). Spindles formed in _cep135_ KO cells displayed an even significantly higher MT density than in controls (Figure 8D). The distribution of \(\gamma\)-tubulin and CDK5 RAP2 ratios between the two spindle poles showed high symmetry (8E and F), in agreement with the observed regular symmetry of spindle structures reflected by comparable MT signals in the two halves of the spindles (Figure 8G). Figure 6: CEP135 expression rescues defects in _cep135_ KO cells. (**A**) Representative immunofluorescence images showing exogenous CEP135 expression in _cep135_ KO cells with antibodies against both N- and C-termini of CEP135. Scale bar: 10 \(\upmu\)m or 2 \(\upmu\)m (insets). (**B**) Centrosomal distance after nocodazole treatment in WT, _cep135_ KO, and rescue cells stably expressing either full-length CEP135, the N-terminus domain of CEP135, or the C-terminus with the centrosomal targeting (PACT) domain of CEP135. (**C**) Quantification of \(\gamma\)-tubulin and CDK5 RAP2 signals in WT, _cep135_ KO, and rescue cells.