The interaction of gremlin-1 with cancer cells is independent of VEGFR2 expression.

<p>(A) Gremlin-1 does not interact with HUVECs as measured by flow cytometry. (B) RT-PCR analysis of VEGFR2 mRNA indicates the presence of VEGFR2 in HUVECs but not in A549 or HeLa cells. (C) Immunoblot analysis using a VEGFR2 antibody indicates that A549 and HeLa cells do not express VEGFR2.</p

Gremlin-1 interacts with human cancer cell lines.

<p>Cells were incubated with gremlin-1, in the presence or absence of the neutralizing antibody GRE1 as described. The four cancer cell lines interacted directly with gremlin-1 and this interaction was inhibited upon the addition of the neutralizing antibody GRE1.</p

Characterization of gremlin-1-transfected A549 cell lines.

<p>(A) RT-PCR and western blot analyses indicate gremlin-1 mRNA and protein are expressed in gremlin-1-A549 cells but not mock-A549 cells. (B) E-cadherin protein expression is reduced in gremlin-1-A549 cells and this effect is attenuated upon addition of the neutralizing antibody GRE1. (C) Gremlin-1-A549 cells show increased invasiveness in cell invasion assays as compared to mock-A549 cells. The cells on the underside of the ECM membrane were stained and counted. ***P<0.001, Student's <i>t</i> test. (D) Gremlin-1-A549 cells show increased migration compared to mock-A549 cells and this effect is attenuated upon the addition of the neutralizing antibody GRE1. **P<0.01, ***P<0.001, Student's <i>t</i> test. (E) Gremlin-1-A549 cells display an increased growth rate compared to mock-A549 cells as determined by MTS proliferation assay. The neutralizing antibody GRE1 addition blocks this effect. *P<0.05 versus mock-A549, Student's <i>t</i> test. (F) Gremlin-1-A549 cells injected subcutaneously in nude mice have an increased rate of tumor growth <i>in vivo</i> as compared with injection of mock-A549 cells. Tumor volume is depicted as the average ± standard deviation. *P<0.05 versus mock-A549, Student's <i>t</i> test.</p

Gremlin-1 induces the scattering and migration of A549 cells <i>in vitro</i>.

<p>(A) A549 cells appear fibroblast-like after incubation with gremlin-1 for 3 days. (B) E-cadherin protein expression is reduced in A549 cells after incubation with gremlin-1 for 3 days. (C) E-cadherin immunofluorescence (green) in A549 cells is reduced after incubation with gremlin-1 for 3 days. Nuclei were counterstained with DAPI (blue) and actin filaments were counterstained with rhodamine-phalloidin (red). (D) Migration of A549 cells after incubation with gremlin-1 only or gremlin-1 plus GRE1. Addition of the neutralizing antibody GRE1 abolishes gremlin-1 induced migration. **P<0.01, ***P<0.001, Student's <i>t</i> test.</p

Addition of the neutralizing antibody GRE1 does not interrupt the interaction between gremlin-1 and BMPs.

<p>(A) Interaction of gremlin-1 with BMP-2, BMP-4, and BMP-7 as measured by enzyme immunoassay. The neutralizing antibody GRE1 does not affect the interaction between gremlin-1 and BMPs. *P<0.05, Student's <i>t</i> test. (B) The interaction of gremlin-1 with A549 cells is unaffected by treatment with a 10 times molar excess of BMP-2, BMP-4, and BMP-7 as measured by flow cytometry.</p