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. Author manuscript; available in PMC: 2015 Apr 30.
Published in final edited form as: Hum Genet. 2014 Apr 20;133(8):1023–1039. doi: 10.1007/s00439-014-1443-3

Figure 6. CENPE-mutated patient LCLs exhibit delayed mitotic progression which is also observed upon conditional expression of CENP-E carrying D933N and K1355E in-cis.

Figure 6

(a). The ratio of binucleates following colcemid block and release into cytochalasin B, compared to untreated (Unt) cells is lower in PCNT-mutated MOPDii (PCNT) or CENPE-mutated patient derived LCLs (LR05-054a2), compared to WT, consistent with a slower progression through mitosis under these conditions in these patient cells.

(b). DLD-1 cells engineered by Flp-In recombination for the conditional (doxycycline-induced) expression of siRNA-resistant CENPE and CENPE patient variants, were plated and 24hrs later subjected to CENPE siRNA targeting endogenous CENP-E only as described in Fig 5a. These cells also contain a RFP-tagged histone H2B to enable real-time analysis of chromosome dynamics during mitosis by live cell imaging. Approximately 40hrs post-siRNA the cells were treated with doxycycline (Dox; 1mg/ml) to induce expression of the siRNA-resistant CENPE variants and cells analyzed by live cell imaging. Still images from the live cell movies following mitosis when wild-type (WT) CENP-E was expressed compared to the double mutant (Double mut) expressing D933N and K1355E in-cis. The time (in minutes) is indicated in the various panels showing onset of the transition from prophase-metaphase-anaphase-telophase under each conditional expression condition.

(c). The time from the onset of nuclear envelope breakdown (NEB) to anaphase was calculated for each CENP-E variant when expressed individually or in-cis (Double mut; double mutant) and represented in box-whisker plots. The average time of onset of NEB to anaphase is shown in red for each variant. Expression of D933N alone, or K1355E alone, did not result in a statistically significant delay compared to expression of wild-type (WT) CENPE (ns; not significant by Student t-test), although the average transition for each variant was slightly longer compared to WT. *Expression of both variants in-cis did result in a statistically significant delay from NEB to anaphase onset compared to WT (p <0.05 Student t-test).