SDC were treated with 50 ng/ml LPS (A) or 100 ng/ml Pam3CSK (B) for the times indicated, then stained for F-actin (red) and vinculin (green)

Arrows indicate focal adhesion–like contacts. The number of focal contacts per cell (C), the percentage of cells with focal contacts (D), and the ventral cell surface area of each cell (E) were quantitated from confocal images of untreated cells and cells treated with 100 ng/ml Pam3CSK for 30 min. Approximately 50 cells for each condition were quantitated for C. At least 450 cells for D and 120 cells for E for each condition, from three experiments performed on independent DC cultures, were scored. Data are means ± SEM. *, P < 0.005. Bars: (A) 10 μm; (B) 20 μm.<p><b>Copyright information:</b></p><p>Taken from "TLR ligand–induced podosome disassembly in dendritic cells is ADAM17 dependent"</p><p></p><p>The Journal of Cell Biology 2008;182(5):993-1005.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528573.</p><p></p

(A) SDC were plated onto thin layers of cross-linked FITC-gelatin for 4 h and fixed, then podosomes were stained with TRITC-phalloidin

(B) xz and yz sections through a z series of confocal images revealed precise coincidence between individual podosomes and holes in the matrix (arrows). (C) BMDC infected with a GFP-actin–expressing retrovirus were plated onto Alexa 594–gelatin, and confocal images were collected every 2 min over a 220-min period. Images from individual time points of Video 2 (available at ) are shown. (D) SDC were treated with 50 ng/ml LPS for 10, 60, or 120 min before plating on FITC-gelatin for a further 2 h. Bars: (A and C) 10 μm; (B) 5 μm; (D) 20 μm.<p><b>Copyright information:</b></p><p>Taken from "TLR ligand–induced podosome disassembly in dendritic cells is ADAM17 dependent"</p><p></p><p>The Journal of Cell Biology 2008;182(5):993-1005.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528573.</p><p></p

(A) BMDC from C3H/HeN and C3H/HeJ mice were labeled with CFSE or CMTMR, mixed in equal numbers with or without 50 ng/ml LPS, and added either into Transwell inserts or directly into control (input) wells

After 2 h at 37°C, migrated or input cells were recovered from the wells, quantitated by flow cytometry, and expressed as ratios of migrated or input HeN/HeJ. (B) DC were pretreated with 100 ng/ml LPS for different lengths of time before addition into the Transwell insert. Note that time = 0* data were obtained from cells where LPS was added immediately before the cells were placed onto the filter. Subsequent migration was for 2 h in all cases. The line indicates the 1:1 ratio achieved where HeN and HeJ migrate with equal efficiency. (C) Data from experiments on three or four independent DC cultures are shown. Bars represent mean data. *, P < 0.05.<p><b>Copyright information:</b></p><p>Taken from "TLR ligand–induced podosome disassembly in dendritic cells is ADAM17 dependent"</p><p></p><p>The Journal of Cell Biology 2008;182(5):993-1005.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528573.</p><p></p

DC from MT1-MMP– (A) and MMP2- or MMP9- (B) deficient mice were fixed after LPS stimulation (50 ng/ml, 30 min) and stained with TRITC-phalloidin to allow assessment of podosome loss

SDC were pretreated for 20 min with 25 μM TAPI-1, 3 μM GW280264X (GW), or 3 μM GI254023X (GI), then stimulated with 100 ng/ml Pam3CSK. TNF-α shedding into the medium measured by ELISA (C) and quantitation of cells with podosomes (D) are shown. Data are representative of experiments performed on DC from at least two mice.<p><b>Copyright information:</b></p><p>Taken from "TLR ligand–induced podosome disassembly in dendritic cells is ADAM17 dependent"</p><p></p><p>The Journal of Cell Biology 2008;182(5):993-1005.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528573.</p><p></p

DC from wild-type or MMP-deficient mice were plated on cross-linked FITC-gelatin matrix for 4 h or for the times indicated, then fixed and stained with TRITC-phalloidin

(A) Quantitation of matrix degradation by DC from MMP2 and MMP9 mice. (B) Confocal images of MT1-MMP and MT1-MMP SDC plated on matrix. Bars 20 μm. (C) Quantitation of matrix degradation by MT1-MMP and MT1-MMP BMDC and SDC. Data are means of experiments performed on DC cultures from two mice of each genotype (error bars indicate the data range), and are representative of data from five MT1-MMP mice in total. (D) The percentage of MT1-MMP and MT1-MMP BMDC and SDC with podosomes when plated on matrix.<p><b>Copyright information:</b></p><p>Taken from "TLR ligand–induced podosome disassembly in dendritic cells is ADAM17 dependent"</p><p></p><p>The Journal of Cell Biology 2008;182(5):993-1005.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528573.</p><p></p

PP1 loss impairs electrotaxis in HeLa cells.

<p><b>A.</b> Treatment of parental HeLa Tet-Off (HTO) cells with siRNA strongly depletes PP1 levels 48 h post transfection. Endogenous PP1 levels were visualized with PP1 antibodies that recognize all isoforms. <b>B.</b> Plot diagrams show that loss of PP1 impairs the ability of cells to migrate towards the cathode. Each line represents the migration trajectory of a single cell. The starting point for each cell migration track is at the origin. Cell tracks with end positions to the right appear in red (“C”, cathode) and those to the left appear in black (“A”, anode). EF-untreated cells were assayed as controls. Control siRNA cells migrate strongly towards the cathode; PP1 siRNA treated cells are unable to migrate in response to a DC EF. Scales show distance migrated in µm. <b>C.</b> PP1 depletion strongly reduces distance migrated, speed, and directedness in response to physiological DC EF. Error bars are S.E.M. <i>p</i> values for significant differences in distance, speed and directedness are shown. <b>D.</b> Localization of endogenous PP1 and distribution of filamentous-actin in control and PP1 depleted cells treated with DC EF. Endogenous PP1 levels were visualized with PP1 antibodies that recognize all isoforms (green) and polymerised actin was detected using rhodamine phalloidin (red). The nuclei have been stained with DAPI (blue). Arrows mark cells with a strong decrease in PP1 levels which correlate with defects in the formation of actin rich protrusions. Representative images are shown. Scale bar is 50 µm. <b>E.</b> Numbers of cells with filopodia were quantified by counting 100 cells. Error bars are S.E.M. <i>p</i> values for significant differences are shown. Images show a detail of cell protrusions in control siRNA and PP1 siRNA cells. Arrows mark numerous filopodia in control cells and outline areas with a major lack of filopodia at the cell edges in PP1 siRNA cells.</p

Cartoon showing the basic organization of the cervical epithelium and a mechanistic model to explain how PP1/NIPP1 may contribute to invasiveness of tumour cells.

<p>Cervical and vaginal epithelia have lumen potentials of about −25 to −50 mV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040769#pone.0040769-Boskey1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040769#pone.0040769-Szatkowski1" target="_blank">[66]</a>. Such a lumen potential would correspond to a transepithelial voltage gradients of 1.7 V/cm (170 mV/mm). In these electrophysiological conditions cervical epithelial cells would migrate towards the lumen as they turn over the epithelial lining layer (green arrow). Upregulation of NIPP1 and its recruitment to PP1 would reverse migration into the lumen, encouraging invasion of the surrounding tissue (red arrow).</p

Effect of pharmacological inhibition of Cdc42-GTPase on the HTO cells.

<p><b>A.</b> Effect of ML141 on Cdc42 GTPase activity in unstimulated cells cultured in complete medium and in EF-stimulated HTO cells overexpressing the FLAG-NIPP1 protein variants. Levels of Cdc42-GTP determined by G-LISA in parental, W.T-NIPP1, ΔC-NIPP1 and mRATA cells in the absence or presence of DC EF and in cells pre-treated with 10 µM of ML141 before electrical stimulation. <i>p</i> values parental to W.T-NIPP1 and parental to ΔC-NIPP1 in complete medium were 0.1 and 0.01, respectively; <i>p</i> values comparing samples in the absence and presence of ML141 were in all cases <0.01. <b>B.</b> Cdc42 inhibition rescues cathodal polarisation and this correlates with centrosome positioning. Directedness values for the migration of EF-treated cells incubated with ML141. Cdc42 inhibition rescues the positive cell directedness decreased by W.T-NIPP1 overexpression. The strongly negative directedness value displayed by ΔC-NIPP1 cells becomes closer to 0 when cells are pretreated with Cdc42 inhibitor. For simplification directedness values in the absence of EF of the parental, W.T-NIPP1, ΔC-NIPP1, and mNIPP1 with and without ML141 have not been included in the diagram. These were, without ML141, −0.07±0.04; 0.05±0.09; −0.08±0.05 and −0.01±0.04, respectively; with ML141 were −0.07±0.04; 0.09±0.05; −0.07±0.05 and −0.01±0.04, respectively. In the absence of EF values were in all cases very close to 0 and differences between the four lines were not statistically significant in any of the cases. Data was quantified from at least three experiments. Error bars are S.E.M. <i>p</i> values for significant differences in directedness are shown. Polarisation index of centrosomes calculated as explained in materials and methods. Polarisation index of W.T-NIPP1 and ΔC-NIPP1 cells becomes similar to the polarisation index of parental cells when cells are treated with the Cdc42 inhibitor ML141.</p

Loss of the PP1 interactor NIPP1 impairs the electrotactic response of PC-3-M cells.

<p><b>A.</b> Treatment of PC-3-M cells with IPTG induces NIPP1 depletion. Cell lysates were analysed by SDS/PAGE and immunoblotting. Bands corresponding to the PP1 isoforms were detected and GAPDH was used as loading control. <b>B.</b> Plot diagrams show that loss of NIPP1 impairs the ability of PC-3-M cells to migrate anodally. Migration trajectories were tracked for three hours. The starting point for each cell migration track is at the origin. Cell tracks with end positions to the right appear in red and those to the left appear in black. Cathode is marked as “C” and anode as “A” when a DC EF is applied to cells. Control scrambled PC-3-M cells migrate strongly anodally (negative directedness value); cells expressing shRNA targeting NIPP1 show a much reduced anodal response. Scales show distance migrated in µm. Scales are different between diagrams in order to include the tracks of every cell assayed. <b>C.</b> NIPP1 depletion strongly reduced distance migrated and directedness in response to physiological DC EF. Data are from at least three experiments. Error bars are S.E.M. <i>p</i> values for significant differences in distance, speed and directedness are shown.</p

List of genes from the Cdc42 pathway that are significantly upregulated by the overexpression of W.T-NIPP1 (WT) or ΔC-NIPP1 (ΔC), but not by mNIPP1 (m), in the HTO cells and compared to parental HTO cells.

<p>List of genes from the Cdc42 pathway that are significantly upregulated by the overexpression of W.T-NIPP1 (WT) or ΔC-NIPP1 (ΔC), but not by mNIPP1 (m), in the HTO cells and compared to parental HTO cells.</p