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. 2015 May 15;26(10):1845–1856. doi: 10.1091/mbc.E14-11-1560

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© 2015 Holland et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Tvwo months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — CENP-E farnesylation is important for chromosome segregation. (A) Experimental approach for the characterization of DLD-1 Flp-In T-Rex cells with integrated RNAi-resistant CENP-E constructs whose expression is inducible with tetracycline. (B) Immunoblot showing successful replacement of endogenous CENP-E with RNAi-resistant MycGFP::CENP-E in DLD-1 cells. C2261S corresponds to the CENP-E farnesylation mutant and T93N to motor-dead “rigor” CENP-E. α-Tubulin was used as a loading control. (C) Immunofluorescence of DLD-1 cells showing loss of kinetochore-localized CENP-E 48 h after transfection with CENP-E siRNA. Cells were costained with ACAs. Scale bar, 5 μm. (D) Quantification of mitotic duration (nuclear envelope breakdown to anaphase onset) from time-lapse fluorescence microscopy experiments conducted with DLD-1 cells expressing histone H2b::mRFP with or without the CENP-E transgenes described in B. Mitotic duration is shown as a scatter plot with the average and SD for the indicated experimental conditions. All conditions were filmed in parallel during the same imaging session using a multiwell chamber. One hundred cells were scored for each condition. The t test was used to determine statistical significance (***p < 0.0001). (E) Quantification of mitotic duration for the indicated conditions from a time-lapse experiment, as described in D (ns, not statistically significant). (F) Selected images from a time-lapse series of DLD-1 cells coexpressing histone H2b::mRFP and CENP-E transgenes showing chromosome congression defects in cells expressing nonfarnesylated CENP-E. Time is indicated in minutes relative to nuclear envelope breakdown (time point 0). Lowercase letters refer to the conditions labeled with the same letters in D and E. Scale bar, 5 μm.