Actin-rich puncta containing cortactin, Arp2/3 complex and N-WASP are apparent in CIA.

<p>(A–C) Cells invading under Matrigel in CIA are fixed and stained with proteins previously localized to invadopodia, cortactin (red), N-WASP (green), Arp2/3 component p34-Arc (green) and actin (blue) in a number of cell lines including MDA-MB-231 (A), CHL1 (B), HT1080 (C). (D) Invadopodia in CIA localize at the front of the invading pseudopods (white arrowhead) and branching sites of the pseudopods (blue arrowhead). Quantification shows there are more invadopodia in the front half of the cells than the rear half. Cells were analyzed in three independent experiments. All error bars indicate means ± SD; **, P<0.01 by Student's t-test. All scale bars 20 µm.</p

Invadopodia structures in CIA have degradation ability.

<p>MDA-MB-231 cells in the CIA assay, showing (A) Cherry-MT1-MMP (red) containing vesicles are delivered to invadopodia structures marked by GFP-N-WASP (green) in CIA. Scale bar 20 µm in the upper panel and 10 µm in the lower panel. (B) DQ collagen is mixed with Matrigel and overlaid on top of the cells in CIA. DQ collagen fluorescence (green) is visualized around some of the actin-rich puncta (blue) with co-localization of N-WASP (red), indicating that they are invadopodia structures in CIA. Scale bar 20 µm.</p

Actin cytoskeletal and focal adhesion organization in MDA-MB-231 cells invading in CIA.

<p>(A) Cells in wound healing assay without Matrigel on 2D surface and cells in CIA with Matrigel overlay were fixed and stained for actin (green), focal adhesion marker phospho-paxillin (red) and DNA (blue). Z-stack confocal images were captured and cell side views are shown to indicate positions of FA/FCs. White arrowheads indicate adhesion complexes. (B) Cells invading in CIA were fixed and stained with actin (red), focal adhesion marker vinculin (green) and DNA (blue). Z-stack confocal images were captured and cell side views are shown to indicate positions of FA/FCs. White arrowheads indicate adhesion complexes. (C) Quantification of adhesion complexes (puncta stained with phospho-paxillin) at the bottom of the cells (within 1 µm range above the glass) and on the cell body or on top of the cells (above 1 µm) under both conditions. All error bars indicate means ± SD; **, P<0.01 by Student's t-test. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030605#pone.0030605.s005" target="_blank">Movies S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030605#pone.0030605.s006" target="_blank">S6</a>.</p

Depletion of Mtss1 affects cell-cell junction maintenance.

<p>Mtss1 siRNA disrupts cell-cell junctions in confluent Scc9 cells. (A) Scc9 cells were treated with siRNA as indicated, NT is a non-targeting siRNA. Boxed region is enlarged on the right. Arrows indicate F-actin structures observed in control cells but not Mtss1 depleted ones. (B) Western blot, representative of 3 experiments showing the Mtss1 depletion with Akt as loading control. (C–E) Normal human keratinocytes treated with siRNA oligos were aggregated in hanging drops. Phase contrast images of single-cell suspension (time zero), and aggregates before (2 h) or after trituration (disaggregation) are shown. (D) Western blot of lysates from treated cells. (E) The area of aggregates following trituration was expressed relative to the original aggregate area (2 h). Values are shown relative to scramble. *** p = 0.001, n = 2.</p

Mtss1 regulates cell-cell junction strength and inhibits HGF-scattering.

<p>(A) Mtss1 construct expression in Scc9 cells. Mtss1-GFP is approximately 3-fold over-expressed vs endogenous Mtss1 (estimated based on densitometry average from 3 experiments). (B) Small colonies of cells were incubated with 10 ng/ml HGF for 20 hours and still photos are shown from live timelapse (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031141#pone.0031141.s003" target="_blank">Movie S1</a>). Graph is % colonies scattering n = 3 experiments. (C) Immunofluorescence labeling of E-cadherin and F-actin following HGF-induced cell scatter for 0, 3 and 6 hours in SCC9 control cells, Mtss1 expressing or K4D mutant expressing as indicated. Images representative of each time point. (D) Effect of HGF treatment on the average number of junctions labeled with E-cadherin per colony is shown relative to Scc9 cells. 40+ colonies were counted per cell line, per experiment (n = 3). (E) Number of single cells released in dispase assay assay (n = 6 experiments). For D&E Mean ± S.E.M. *** p<0.01, ** p, 0.05 by T-test.</p

Mtss1 enhances de novo cell-cell junction formation.

<p>(A, B) Scc9 cells +/− Mtss1-GFP were treated with 2 mM EGTA for 20 min, to disrupt junctions. Reassembly was stimulated by 2 mM Ca²⁺ and cells were labeled for E-cadherin and F-actin. E-cadherin and F-actin were visualized following triton extraction and fixation to preserve the triton-insoluble junctional cytoskeleton. (A) Cell-cell junction formation in Scc9 cells and (B) Mtss1-GFP expressing Scc9 cells (C) Mean intensity of E-cadherin fluorescence at cell-cell junctions in Scc9 cells ± Mtss1 (mean ± S.D) from 3 independent experiments where n = 20 junctions. (D) Scc9 cells ± Mtss1-GFP cultured in low-Ca²⁺ KSFM overnight to disassemble adherens junctions. Cells were Ca²⁺ treated, fixed and labeled after the indicated times. Enlarged images below show boxed regions.</p

Primitive fetal liver cells from Rosa26-tdRFP⁺;Pf4-Cre⁺ mice express RFP.

<p>Fetal liver from day E14.5 Rosa26-tdRFP⁺;Pf4-Cre⁺ and litter matched controls was isolated and (<b>A</b>) the Sca-1⁻c-kit⁺ cells identified in the Lin⁻ cell compartment. LK compartment was subfractionated using side scatter (SSC) and RFP expression to identify the total number of RFP⁻ (black) and RFP⁺ (red) cells. Dot plots are representative figures of three independent experiments with the average±SEM from three independent experiments. (<b>B</b>) RFP expression in HSCs and primitive progenitors within the LSK compartment. The LSK cell compartment was subfractionated using CD48 and CD150 to identify the frequencies of RFP⁺ (red) in LSK CD150⁺CD48⁻ HSC (Compartment I), LSK CD150⁻CD48⁻ (Compartment II), LSK CD150⁺CD48⁺ (Compartment III) and LSK CD150⁻CD48⁺ (Compartment IV) compartments. Dot plots and histograms are representative figures of three independent experiments with the mean±SEM from three independent experiments.</p

Pf4-Cre activates RFP expression in all hematopoietic lineages.

<p>(<b>A</b>) Frequencies of RFP-expressing B-lymphoid (CD19⁺), myeloid (Gr-1⁺), and erythroid (Ter119⁺) cells in the bone marrow obtained from adult Rosa26-tdRFP⁺;Pf4-Cre⁺ mice and Rosa26-tdRFP⁺;Pf4-Cre⁻ litter matched controls. (<b>B</b>) Frequencies of RFP-expressing B-lymphoid (CD19⁺), myeloid (Gr-1⁺), and erythroid (Ter119⁺) cells in the spleen from adult Rosa26-tdRFP⁺;Pf4-Cre⁺ mice and Rosa26-tdRFP⁺;Pf4-Cre⁻ litter matched controls. (<b>C</b>) Frequencies of RFP-expressing CD4⁺ T cells in the spleen and thymus of adult Rosa26-tdRFP⁺;Pf4-Cre⁺ mice and Rosa26-tdRFP⁺;Pf4-Cre⁻ litter matched controls. Dot plots shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051361#pone-0051361-g001" target="_blank">Figure 1A–C</a> are representative figures of 3–5 independent experiments with values shown as an average±SEM from 3–5 independent experiments. % of RFP⁺ cells is indicated in red and % of RFP⁻ in black. (<b>D</b>) Bone marrow from Rosa26-tdRFP⁺;Pf4-Cre⁺ and litter matched controls was fixed, and histologically stained for RFP. Images are representative of 2 independent experiments.</p

Pf4 gene expression is identified in Stem and primitive progenitor cell populations.

<p>Pf4 expression in LSK CD150⁺CD48⁻ HSCs, LSK CD150⁻CD48⁻, LSK CD150⁺CD48⁺, and LSK CD150⁻CD48⁺ cells sorted from bone marrow of control mice. Relative Pf4 expression levels were calculated with the LT-HSC fraction set to the value of 1 (see methods). Results are mean±SD from three independent experiments.</p

Pf4-Cre genetically marks the adult stem cell and early progenitor cell compartment.

<p>Bone marrow from Rosa26-tdRFP⁺;Pf4-Cre⁺ and litter matched control mice was isolated and (<b>A</b>) the Sca-1⁻c-kit⁺ cells identified in the Lin⁻ cell compartment. LK compartment was subfractionated using side scatter (SSC) and RFP expression to identify the total number of RFP⁻ (black) and RFP⁺ (red) cells. (<b>B</b>) The LK population was subfractionated using CD34 and CD16/32 to identify RFP⁺ (red) cells in the megakaryocyte-erythrocyte progenitor (MEP) (Compartment I), common myeloid progenitor (CMP) (Compartment II) and granulocyte-macrophage progenitor (GMP) (Compartment III) cell populations. (<b>C</b>) RFP expression in HSCs and primitive progenitors in the LSK compartment. The LSK cell compartment was subfractionated using CD48 and CD150 to identify the frequencies of RFP⁺ (red) cells in LSK CD150⁺CD48⁻ HSC (Compartment I), LSK CD150⁻CD48⁻ (Compartment II), LSK CD150⁺CD48⁺ (Compartment III) and LSK CD150⁻CD48⁺ (Compartment IV) compartments. Dot plots and histograms are representative figures of three independent experiments with the mean±SEM from three independent experiments.</p