Fig. 2: INPP5E accumulates at the immune synapse in activated T cells.

a Jurkat cells were transfected with either control (siCtrl) or INPP5E-specific siRNA (siINPP5E). INPP5E expression was analyzed by immunoblotting. Gel bands were quantified. Error bars indicate mean ± SD. N = 5 by student T-test. ****P < 0.0001. b Jurkat cells were transfected with either siCtrl or siINPP5E. SEE-coated Raji cells conjugated to Jurkat T cells were fixed and immunostained for INPP5E. Scale bar: 10 μm. c In the left panel, the percentage of INPP5E recruitment in conjugates at immune synapses from four independent experiments. n = 122 conjugates for siCtrl, and n = 111 conjugates for siINPP5E. In the right panel, the recruitment index of INPP5E was quantified. n = 39 for siCtrl and n = 35 for siINPP5E. Error bars indicate mean ± SD. Unpaired student T-test. **P < 0.005. ****P < 0.0001 d Jurkat cells were transfected with either control or siINPP5E. Representative TIRF images of INPP5E and CD3ζ in spreading Jurkat T cells on anti-CD3/CD28-coated coverslips for 10 min are shown. Scale bar: 5 μm. A schematic figure of the spreading assay is shown. e The mean fluorescence intensity (MFI) of INPP5E and CD3ζ from (d) was quantified. Data were obtained from three independent experiments. n = 24 cells for siCtrl, and n = 25 cells for siINPP5E. Unpaired student T-test. **P < 0.005. ****P < 0.0001. f Flag-INPP5E was expressed in conjugates of Jurkat T cells and CMAC-labeled SEE-pulsed APCs, followed by immunostaining. Cells were co-stained with an anti-CD3ε antibody as the immune synapse marker. Scale bar: 10 μm. g The recruitment of INPP5E at immune synapses from (f) was quantified. N = 4. n = 21 conjugates for -SEE, and n = 26 conjugates for +SEE. Unpaired student T-test. ****P < 0.0001. h Immunostaining of INPP5E in T-cell-APC conjugates with different time points. Scale bar: 10 μm. i Quantification of the INPP5E recruitment index (upper panel) and the INPP5E enrichment events (lower panel) in conjugates were from two independent experiments. n = 34 for 2 min, n = 70 for 5 min, and n = 63 for 10 min. 0 min represent an experiment where cells were fixed before adding APCs. Error bars indicate mean ± SD. One-way ANOVA analysis. *P ≤ 0.05. **P ≤ 0.005. ***P < 0.001. ****P < 0.0001. j Three-dimensional structured illumination microscopy (3D-SIM) images of INPP5E in T-cell APC conjugating at different time points. CD45 was used as a T-cell surface marker. Enlarged figures are shown on the right. The numbers 1-3 indicate z-stack intervals from the top of each cropped 3D-SIM image. Scale bars are as indicated.