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

Figure 5. Aurora A inhibition does not affect actin cytoskeleton dynamics.

Figure 5

(a) Immunoblot of a pull-down assay of GST-Nck fusion protein from cell lysates of control (DMSO; vehicle) or Aurora A inhibitor (MLN8237)-pretreated human T lymphoblasts. Activation was performed with soluble anti-CD3ɛ antibodies for indicated times. CD3ζ and GST are shown. CD3ζ content in whole-cell lysates is indicated in the bottom row. (b) Immunoblotting of Rac1 pull-down assay of GST and GST-PAK-CD from cell lysates of DMSO- or MLN8237-pretreated Jurkat T cells activated with SEE-pulsed Raji B cells (APCs) for the indicated times. Loading control for Rac1 in whole-cell lysates is shown. (c) Images from TIRFm time-lapse analysis of mCherry–β-actin-expressing Jurkat T cells spreading over anti-CD3/CD28-coated glass-bottom chambers. Cells were pretreated with DMSO or MLN8237. Images were taken every 100 ms for 5 min at 90 nm penetrance. A corresponding bright-field image is shown. Scale bar, 10 μm. (d) Quantification of the area occupied by the whole cell (lamella), the actin-rich area (peripheral SMAC (pSMAC)), the central area (cSMAC) and the distribution of mean fluorescence intensity per area (ratios cSMAC:cell; pSMAC:cell and cSMAC/pSMAC) from cells in c (n=48 and n=36, three independent experiments). Cells were fixed after spreading (4 min) and fluorescence images were taken. Data represent means±s.d.; t-test. n.s., nonsignificant. (e) Maximum Z projections of confocal stacks from DMSO- or MLN8237-pretreated Jurkat T cells conjugated with SEE-APCs. Cells were incubated for 30 min, fixed and stained for α-tubulin (green) or actin (magenta). The right-hand image shows CMAC cell tracker labelling of APCs (cyan) and bright field. Scale bar, 10 μm. (f) Quantification of actin accumulation at the IS contact area in conjugates as in e from three independent experiments (n=100). Data represent means±s.d.; t-test. (g) Image sequence for IS formation between mCherry–β-actin-expressing T cells and SEB-APCs (DMSO- or MLN8237-treated). XYZ stacks were acquired every 25 s (maximal projections of XYZ stacks and 3D reconstructions with Imaris Software are shown from representative conjugates). (h) Ratio of T cells forming lamella on contact with an APC from g. Data represent median±interquartile range. Mann–Whitney test (DMSO: 28 cells (n=4); MLN8237: 25 cells (n=3)).