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

Figure 1. Aurora A is located at the IS contact area and is activated on TCR triggering.

Figure 1

(a) Maximum Z projection of a confocal stack of human primary CD4+ T cells pretreated with vehicle (DMSO) or Aurora A inhibitor (MLN8237, 10 μM) and conjugated with anti CD3/CD28-coated beads. Images show three representative conjugates in DMSO and two in MLN8237-treated cells at different stages of cell conjugation. Cells were fixed and stained for PKCθ (red), T288-phosphorylated Aurora A (magenta) and α-tubulin–fluorescein isothiocyanate (FITC) (green). Bright field with DAPI frames are included. Scale bar, 10 μm. (b) Quantification of T288-phosphorylated Aurora A accumulation at the IS contact area in conjugates as in a from three independent experiments (n=93 in non-activated, n=105 in DMSO, n=109 in MLN8237). Data represent means±s.d. Means were compared with a t-test. (c) Maximum Z projections of confocal stacks of transgenic OTII CD4+ cells conjugated with OVA peptide-pulsed bone-marrow-derived dendritic cells (DCs). Cells were incubated for 30 min, fixed and immunostained for T288-phosphorylated Aurora A (magenta) and actin (green). The right-hand image shows CMAC cell tracker labelling of DCs (cyan) and bright field. Scale bar, 10 μm. (d) Maximum Z projection of a confocal stack of human primary CD4+ T cells transfected with Aurora A-GFP WT or Aurora A-GFP KD (green) and conjugated with anti CD3/CD28-coated beads. Cells were incubated for 30 min, fixed and stained for T288-phosphorylated Aurora A (magenta). Bright field with DAPI frames are included. Scale bar, 10 μm. (e) Quantification of T288-phosphorylated Aurora A and transfected Aurora A accumulation at the IS contact area in conjugates as in d (n=45 in Aurora A-GFP WT, n=29 in Aurora A-GFP KD). Data represent means±s.d. Means were compared with a t-test. n.s., nonsignificant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.