Fig. 3. APC silencing impairs migration and adhesion of CEM T cells.

CEM T cells were transfected with control (siCTRL) or APC (siAPC) siRNA oligonucleotides and used 72 hours after transfection. (A) Western blot showing the expression level of APC protein in siCTRL- and siAPC-transfected CEM T cells. A representative result is shown in the left panel. APC band intensity was quantified from four independent immunoblots and normalized by the SLP76 intensity in the same sample (right panel). (B) Transmigration through transwell filters was analyzed for control and APC-silenced CEM T cells. Bar plots represent the mean + SD of T cell–specific migration toward CXCL12. (C, F, and G) Surface expression of the CXCL12 chemokine receptor CXCR4 (C) and the α₄β₁/VLA-4 (F) and α_Lβ₂/LFA-1 (G) integrins was measured by flow cytometry. Boxes display the median fluorescence intensity. (D and E) siCTRL- and siAPC-CEM T cells were seeded in VCAM-1 or fibronectin or ICAM-1 + CXCL12–coated chambers, and a laminar shear flow of PBS was applied through the chamber (movie S3). (D) The number of adherent cells per frame was plotted as a function of the force on the different substrates. (E) Boxes represent the rupture force of adhesion to VCAM-1–, fibronectin-, and ICAM-1 + CXCL12–coated surfaces of siCTRL versus siAPC T cells. (H and I) VLA-4 and LFA-1 integrin activation was assessed by measuring the binding of T cells with the corresponding integrin ligands: VCAM-1 (H) and ICAM-1 (I). Boxes display the median fluorescence intensity. (A to I) Statistical differences (more than four independent experiments) were calculated by Mann-Whitney unpaired test. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05; ns, not significant.