Figure 4. CENPE-mutated patient LCLs exhibit abnormal spindle microtubule organization and evidence of aberrant segregation and delayed progression through mitosis.

(a). An image of a lagging chromosome in an anaphase cells from an asynchronous culture of LR05-054a1.

(b). The lower panels show IF images of α-tubulin staining microtubule network (green) from wild-type (WT) and LR05-054a1 LCLs following treatment with Taxol (10µM) and MG132 (10µM) for 1hrs then 1.5hrs on ice to stabilize spindle microtubules. The upper panels are the corresponding DAPI-staining nuclei. A normal bipolar spindle is seen in WT mitotic cell whilst multipolar spindles are frequently seen in mitotics from LR05-054a1.

(c). The distribution of spindle abnormalities noted in mitotic cells in wild-type (WT), PCNT-mutated MOPDii (PCNT) and CENPE-mutated patient derived LCLs (LR05-054a1). Between 40–50 mitotic cells were scored for each cell line. Multipolar spindles are over-represented in mitotic cells from PCNT and LR05-054a1 LCLs compared to WT.

(d). The spontaneous levels of micronuclei (MN) are not statistically significantly elevated (ns; not significant Student t-test) in PCNT-mutated MOPDii (PCNT) or CENPE-mutated patient derived LCLs (LR05-054a2) compared to WT. In contrast, binucleates (BN) levels are approximately 2-fold higher in these LCLs compared to WT potentially suggestive of elevated rates of cytokinesis failure (* p<0.05 Student t-test).

(e). DAPI (upper panel) and their corresponding IF-mediated β-tubulin images (lower panel) of binucleates show that the binucleates (circled) from LR05-054a2 LCLs frequently exhibit nuclei of different sizes. The β-tubulin staining facilitates the identification of true binucleate cells. Differing sized nuclei within a binucleate are indicative of unequal segregation during mitosis.