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1.
Figure 4

Figure 4. ERM proteins are required for uropod formation in response to β1, but not β2 integrin engagement.. From: Ezrin and Moesin Are Required for Efficient T Cell Adhesion and Homing to Lymphoid Organs.

(A) Wild-type or ERM-deficient T cells were allowed to interact with fibronectin-coated glass coverslips, fixed and imaged using DIC optics. Uropods are marked with arrows. (B) Quantitation of uropod formation in cells analyzed in (A). (C) Wild-type or ERM-deficient T cells were allowed to interact with glass coverslips coated with anti-human IgG followed by rVCAM-1 Fc and imaged as in (A). (D) Quantitation of uropod formation in cells analyzed in (C). (E) Wild-type or ERM-deficient T cells were allowed to interact with glass coverslips coated with anti-human IgG alone (top panels) or together with rICAM-1 Fc (bottom panels), and imaged as in A. (F) Quantitation of uropod formation in cells analyzed in (E). Data in B, D and F represent mean ± StDev of at least 5 coverslips, with 50–100 cells each, from 2–7 independent experiments. *p<0.05, **p<0.005.

Emily J. H. Chen, et al. PLoS One. 2013;8(2):e52368.
2.
Figure 3

Figure 3. ERM proteins are required for efficient adhesion to β1, but not β2 integrin ligands.. From: Ezrin and Moesin Are Required for Efficient T Cell Adhesion and Homing to Lymphoid Organs.

(A and C) Wild-type or the indicated ERM-deficient T cells were stained with Calcein-AM and settled in 96-well plates coated with fibronectin (A) or rICAM-1 Fc (C). Cells were either left unstimulated or stimulated as indicated, non-adherent cells were washed off, and fluorescence was measured using a microplate reader. Adherent cells are shown as a percentage of input. Data shown are means ± StDev from triplicate wells in one experiment, representative of three individual experiments. * p<0.05, **p<0.005. (B and D) Cells were stained with either anti-CD29 (B) or anti-CD18 (D) and analyzed by flow cytometry to assess surface integrin levels.

Emily J. H. Chen, et al. PLoS One. 2013;8(2):e52368.
3.
Figure 2

Figure 2. ERM-deficient T cells can migrate efficiently in confined spaces.. From: Ezrin and Moesin Are Required for Efficient T Cell Adhesion and Homing to Lymphoid Organs.

A) Wild-type or ERM-deficient T cells were placed in a 5 µm pore collagen gel in the absence (top panels) or presence (bottom panels) of a CCL19 gradient, and cell migration was imaged for 4 hours at 37°C. Tracks of individual cells are presented with the same point of origin. Data are representative of three experiments. Quantitative analysis is presented in Tables 1 and 2. B and C) Frequency of individual cell velocities in (A) in the absence (B) or presence (C) of chemokine. Data are representative of at least three collagen gels per condition in two independent experiments.

Emily J. H. Chen, et al. PLoS One. 2013;8(2):e52368.
4.
Figure 1

Figure 1. ERM-deficient T cells can chemotax efficiently in vitro.. From: Ezrin and Moesin Are Required for Efficient T Cell Adhesion and Homing to Lymphoid Organs.

Wild-type T cells expressing both ezrin and moesin (Ez+/+MoSiC), T cells lacking moesin (Ez+/+MoSiM), T cells lacking ezrin (Ez−/−MoSiC), or T cells lacking both ezrin and moesin (Ez−/−MoSiM), were prepared as described in Materials and Methods. (A) Cells were stained for CCR7 and assessed by flow cytometry. Filled histogram, isotype control. (B–D) Wild-type or the indicated ERM-deficient T cells were placed in a transwell assay in the absence or presence of 40 nM CCL19 for 2 hours. Cells that migrated across a 5 µm (B) or 3 µm (C and D) pore membrane to the bottom well were quantified, and are presented as the percentage of input. Data are mean ± StDev of quadruplicate wells from one experiment, representative of three experiments. *p<0.05, **p<0.005.

Emily J. H. Chen, et al. PLoS One. 2013;8(2):e52368.
5.
Figure 5

Figure 5. ERM proteins are required for T cell homing to peripheral lymphoid organs.. From: Ezrin and Moesin Are Required for Efficient T Cell Adhesion and Homing to Lymphoid Organs.

Wild-type and ERM-deficient T cells were differentially labeled with CFSE and CMTMR, mixed in a 1∶1 ratio and injected into the tail veins of C57Bl/6 hosts. (A) Blood, spleen, and peripheral and mesenteric lymph nodes were collected 1 hour after injection, and cell suspensions were analyzed by flow cytometry. The ratio of adoptively transferred ERM-deficient to wild type T cells is shown. Data represent mean ± StDev from 5 mice in one experiment, representative of four experiments. * p<0.05, **p<0.005. (B and C) Lymph nodes were harvested 1 hour after injection for multi-photon imaging, and cell migration was tracked. (B) The percentage of cells showing active migration, defined as detailed in Materials and Methods. (C) Tracks, average velocities and average track lengths from one representative lymph node. Data represent mean ± StDev of three experiments.

Emily J. H. Chen, et al. PLoS One. 2013;8(2):e52368.
6.
Figure 6

Figure 6. ERM-deficient cells undergo efficient diapedesis in vitro.. From: Ezrin and Moesin Are Required for Efficient T Cell Adhesion and Homing to Lymphoid Organs.

(A,B) Diapedesis under static conditions. Confluent monolayers of 3B-11 endothelial cells were pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces. Cells were imaged in an environmental chamber every 30 seconds for 2 hours. (A) DIC images of cells undergoing diapedesis. Arrowheads indicate leading edges of migrating T cells. White arrows indicate cells migrating along the apical surface of endothelial cells; the same cells are marked with red arrows after passing below the endothelial cell layer. (B) Quantitative analysis of assays carried out as in A. Cells undergoing diapedesis are quantified as a percentage of total moving cells. Data represent mean ± StDev from six experiments. (C) Diapedesis under shear flow conditions. Confluent monolayers of 3B-11 endothelial cells were grown in flow chambers, pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces and allowed to interact with endothelial cells under shear stress of 0.5 dyne/cm2. Cells were imaged every 30 seconds for 1 hour, and the percentage of moving cells that underwent diapedesis was determined. Data represent mean ± StDev from three experiments.

Emily J. H. Chen, et al. PLoS One. 2013;8(2):e52368.

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