Extended Data Fig. 11: Thromboxane A2 induces PTEN recruitment and suppresses AKT phosphorylation in T cells via ROCK kinase.
From: Aspirin prevents metastasis by limiting platelet TXA2 suppression of T cell immunity
a, Confocal immunofluorescence imaging of WT and Arhgef1-KO OT-1 CD8 T-cells 5 days after primary stimulation and subsequently seeded on anti-CD3 antibody-coated coverslips with or without 10 min of stimulation with U-46619 (10 μM) and/or 1 h of preincubation with the TP inhibitor (SQ 29548, 10 μM) or ROCK inhibitor (Y-27632, 30 μM). Images were captured on a Leica confocal microscope using a 63x oil objective lens, in three channels, F-actin (yellow) and PTEN (red) plus a DAPI nuclear counter stain (blue) to locate the cells. Images analysed were maximum intensity projections of four consecutive z-slices selected from the centre of a cross-volume image stack. b, Immunoblot analysis of protein phosphorylation pre-activated (day 5) WT or Arhgef1 KO OT-1 TCR transgenic CD8+ T cells stimulated in vitro with crosslinked anti-CD3 antibodies for 5 min in the presence of TXA2 analogue or vehicle control and treatments with the PTEN inhibitor (bpV(pic), 2.5 μM) or ROCK inhibitor (GSK269962A, 10 μM). Data are representative of two independent experiments.
