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. 2016 Nov 7;215(3):383–399. doi: 10.1083/jcb.201603080

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Figure 7. — Functional consequences of formin inhibition. (A) 3D-SIM images of phalloidin-stained (grayscale) DMSO-treated or 10 µM SMIFH2-treated Jurkat T cells stimulated on planar lipid bilayers containing Alexa Fluor 647–labeled anti-CD3 (red) to report TCR MC distribution. (B) Same as in A, except cells were stained with M24 antibody (green) during IS formation to determine the distribution of open, active LFA-1. Radial plot profiles of TCR MCs (C) and LFA-1 clusters (D) after DMSO or SMIFH2 treatment. n = 14–16 cells/condition; Data are means ± SEM; P < 0.0001, two-way ANOVA. (E) Line scans across cells in B fit to Gaussian distributions. (F) Adhesion conjugate assay after DMSO, SMIFH2, or pnBB treatment. n ≥ 3 independent experiments; data are means ± SEM. (G) Combination masking strategy to quantitate fluorescence at the T cell IS. (H) Imaging flow cytometry of Jurkat T–B cell conjugates fixed, immunostained, and analyzed on a flow cytometer for background-corrected MFI (Norm. Fluor. Int.) of the indicated signaling proteins after DMSO, SMIFH2, or pnBB treatment. n ≥ 3 independent experiments; data are means ± SEM. Note that the differences remain statistically significant even when background correction is omitted (DNS). Bars, 5 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. a.u., arbitrary units; Norm. Fluor. Int., normalized fluorescence intensity.