Characteristics of the secondary antibodies used in the study.

<p>(Abbreviations: IH = immunohistochemistry, IF = Immunofluorescence, WB = Western blot, IP = Immunoprecipitation).</p

Effect of the down regulation of IQGAP1 expression on F- actin network.

<p>(A) IQGAP1 Western blot analysis showing IQGAP1 expression in podocytes transfected with siRNA IQGAP1 as compared with untransfected podocytes (UT) or podocytes transfected with siRNA directed at luciferase (Luc). (B) F-actin labelling after siRNA IQGAP1 transfection in comparison to control cells (untransfected cells). Scale bars, 50 µm.</p

Cellular localization of interactions between IQGAP1 and different proteins of the slit diaphragm: In situ Proximity Ligation assay.

<p>In situ Proximity Ligation assays confirmed in cells the interactions between IQGAP1 and nephrin, MAGI-1, CD2AP, podocin, NCK1/2 and podocalyxin. Cells were transfected with siRNA Luc (Luc) or siRNA IQGAP1 (siRNA).</p

Involvement of IQGAP1 in podocyte proliferation, viability and adhesion.

<p>Influence of down regulation of IQGAP1 on cell proliferation and viability (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037695#pone-0037695-g007" target="_blank">Figure 7A and 7B</a>, n = 5, Two-way repeated measurements ANOVA). Cell survival was not altered by transfection. Increase of cytotoxicity was not the course of the migration defect. Podocyte adhesion after down regulation of IQGAP1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037695#pone-0037695-g007" target="_blank">Figure 7C</a>, n = 3, Wilcoxon test). Adhesive properties of podocytes were conserved after siRNA IQGAP1 transfection. Control: control cells; siRNA: siRNA IQGAP1 transfected podocytes.</p

siRNA used in the study.

<p>siRNA used in the study.</p

IQGAP1 glomerular expression and localization in normal kidney tissue.

<p>In normal glomeruli, IQGAP1 labelling (green, Panel A, a) was continuous, corresponding to podocytes (plain and fine arrows →), as was nephrin staining (red, Panel B and b, dashed arrow ? ). The expression in parietal epithelial, endothelial and distal tubular cells in normal glomeruli is shown respectively by bold arrows in Panel A and C. Panel C showed the co-expression of IQGAP1 and nephrin. Scale bars, 50 µm. Panels a, b and c are enlarged images of the boxed areas in panels A, B and C respectively. Results shown are representative of paraffin kidney sections from 7 normal kidneys (Results of the 6 other kidneys are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037695#pone.0037695.s005" target="_blank">Figure S5</a>). Scale bar, 50 µm. Analysis was performed with a confocal microscope (Leica). Microscope sections, 0.5 µm. Magnification, X40.</p

Western blot analysis of IQGAP1 expression in cultured podocyte extracts.

<p>(A) Western blot analysis of IQGAP1, detected at a molecular weight of 180 kD, and β-actin expressions during podocyte differentiation (top panel). Protein extracts were from undifferentiated (permissive temperature of 33°C, Day 0 (D0)) and differentiated (non-permissive temperature of 37°C, Day 16 (D16)) cultured immortalized podocytes. HKL: Human Kidney Lysate; MW: Molecular weight (kDa). The blot is representative of six independent experiments. (B) IQGAP1 expression at undifferentiated and differentiated stages. IQGAP1 expression, determined by densitometry, was plotted and reported to β-actin expression. Data are representative of six independent experiments. <i>Asterisk,</i> significantly different from undifferentiated cells (p<0.01, Wilcoxon’s test). (C) Western blot analysis of nephrin, MAGI-1, CD2AP, podocin, NCK 1/2, α-actinin-4, β-catenin, podocalyxin and β-actin during podocyte differentiation.</p

Involvement of IQGAP1 in podocyte migration.

<p>To assess the role of IQGAP1 in cell migration, migrations assay were performed with control cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037695#pone-0037695-g006" target="_blank">Figure 6A and B</a>) compared to siRNA IQGAP1 transfected cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037695#pone-0037695-g006" target="_blank">Figure 6C and D</a>). Cell migration was evaluated during 48 hours with sequential images acquisitions. Wound closure was expressed as the percentage of cellularized surface (data are representative of 5 independent experiments, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037695#pone-0037695-g006" target="_blank">Figure 6E</a>). Control: control cells; siRNA: siRNA IQGAP1 transfected podocytes.</p

Subcellular localization of IQGAP1 and slit diaphragm proteins or podocalyxin.

<p>Double immunolabelling experiments were performed on cultured differentiated podocytes, and analyzed by confocal microscopy. Column 1: IQGAP1 (red). Column 2: nephrin (a), MAGI-1 (b), CD2AP (c), podocin (d), NCK 1/2 (e), α -actinin-4 (f), β-catenin (g), β-actin (h), F-actin (i) and podocalyxin (j) (green). Column 3: Colocalization of IQGAP1 and studied proteins, observed as a merge signal in yellow staining or as colocalization points in black color (Column 4) with the Leica analysis software.</p

IQGAP1 interactions with proteins of the slit diaphragm and podocalyxin.

<p>IQGAP1 immunoprecipitation experiments were performed with undifferentiated podocytes (D0, 33°C) and differentiated podocytes (D16, 37°C), using equal amounts of total protein. The irrelevant antibody against tristetraprolin was used in control immunoprecipitations (Ctl). (A) Interaction between IQGAP1 and nephrin (data are representative of 6 independent experiments). (B to H) Interactions between IQGAP1 and MAGI-1, CD2AP, podocin, NCK1/2, α-actinin-4, β-catenin and podocalyxin (Data are representative of 5 independent experiments, 6 for podocalyxin).</p