Kindlin-3 regulates the integrin αMβ2-Syk-Vav1 signaling axis.

<p>KM, ctrl-KM and k3-KM cells were seeded into iC3b-coated TC dishes in the presence of irrelevant mouse IgG (IgG) or mAb KIM185 (10 µg/ml each) and incubated under culture conditions for 30 min. Cells were harvested and lysed followed by immunoprecipitation (IP) using either anti-Syk or anti-Vav1 antibody with mouse IgG or rabbit anti-GST antibody as irrelevant IgG, respectively. Tyr-phosphorylated Syk and Vav1 were probed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056911#s2" target="_blank">materials and methods</a>. IB: immunoblotting. A representative experiment from two independent experiments is shown.</p

Kindlin-3 is required for integrin αMβ2-mediated cell spreading.

<p>ECIS measurements of ctrl-KM and k3-KM cells spreading on iC3b or BSA. Each data point represents the mean ± SD of technical triplicates at 1 min intervals. mAbs LPM19c and KIM185 were used at 10 µg/ml each. A plot of a representative experiment from three independent experiments is shown for each ligand.</p

Knockdown of kindlin-3 expression in K562 cells expressing integrin αMβ2.

<p>(A) qPCR analyses of kindlin-3 mRNA expression level in ctrl-KM and k3-KM cells. (B) Expression levels of kindlin-3 and other proteins in these cells were determined by immunoblotting. Actin was used as loading control. (C) Cell surface expression of integrin αMβ2 was determined by flow cytometry. Shaded and open histograms represent control IgG and mAb LPM19c stainings, respectively. GP: gated positive; GM: geo-mean; EI: expression index. (D) To determine extracellular activation of integrin αMβ2 on ctrl-KM and k3-KM cells. Cells were treated with Mn²⁺ (1 mM) or without and stained with mAb KIM127 at 37°C. Control IgG (ctrl-IgG) and mAb LPM19c were included for each condition. The %GP, GM and EI of mAb KIM127 staining are shown. One representative experiment out of two independent experiments is shown.</p

Integrin αMβ2-induced RhoGTPase activation involves kindlin-3.

<p>Ctrl-KM and k3-KM cells were allowed to adhere to iC3b-coated TC dishes in the presence of mAb KIM185 (10 µg/ml). (A), (C) and (E) are immunoblots of cell lysates for Rac1, Cdc42 and RhoA, respectively. (B), (D) and (F) are pull-down experiments using cell lysates and RBD or PBD-conjugated beads. IB: immunoblotting. A representative experiment from two independent experiments is shown.</p

Reduced kindlin-3 expression diminished integrin αMβ2-mediated cell adhesion.

<p>(A) and (B) show adhesion data of ctrl-KM and k3-KM cells on iC3b and BSA, respectively. Each data point represents the mean ± SD of three independent experiments. mAbs LPM19c and KIM185 were used at 10 µg/ml each. (C) Shear flow analyses of ctrl-KM and k3-KM cells in flow chambers coated with iC3b. Each data point is the mean ± SD of number of cells in four fields and a representative plot of two independent experiments is shown.</p

Kindlin-3 is involved in integrin αMβ2 outside-in signaling.

<p>(A) Flow cytometry analyses of αMβ2N329S in cells transduced with control or kindlin-3-targeting siRNA. Shaded and open histograms represent control IgG and LPM19c stainings, respectively. GP: gated positive; GM: geo-mean; EI: expression index. (B) Cell adhesion assay on iC3b. (C) ECIS measurements on iC3b. In (B) and (C), each data point represents mean ± SD of technical triplicates. mAb LPM19c was used at 10 µg/ml. (A–C), a single representative experiment from three independent experiments is shown.</p