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. 2016 Apr 19;7:11389. doi: 10.1038/ncomms11389

Figure 3. Aurora A gene ablation impairs MT dynamics at the IS.

Figure 3

(a) Immunoblot analysis of Aurora A protein expression in CD4+ T cells WT and KO. (b,c) Imaging of EB3-GFP-expressing Aurora-A-deficient and control CD4+ T cells, pretreated with DMSO or MLN8237 and settled on corresponding anti-CD3/CD28-coated glass-bottom chambers. Maximal projection of XYZ stacks for fluorescence and single bright-field (BF) images are shown. Scale bar, 5 μm. (c) Ratio of EB3-GFP fluorescence incorporated in +tips from XYZ stack (0 s, n=25 in WT, n=17 in KO, n=22 in WT MLN8237 and n=17 in KO MLN823). Data represent means±s.d. Means were compared with a Mann–Whitney test. Map of the trajectories of EB3-GFP-decorated MT plus tips in human CH7C17 T cells pretreated with DMSO or MLN8237 (d,e), or in Aurora-A-deficient and control CD4+ T cells (f,g) and settled on anti-CD3/CD28-coated glass-bottom chambers. Images were taken every 300 ms under a TIRF microscope at a penetrance of 150 nm. MT +tips were tracked with Imaris software over 5 (d) or 2 min (f). Maximal projections of the time lapse from representative cells are shown. Scale bar, 10 μm. (e,g) Quantification of the number of MT plus tip tracks presented in d (e; n=7 in DMSO, n=9 in MLN8237) and g (f; n=6). Error bars represent interquartile rage. Medians were compared with a Mann–Whitney test. n.s., nonsignificant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.