Journal: Oncogene

Article Title: Ligand independent activation of c-Met by fibronectin and α 5 β 1 -integrin regulates ovarian cancer invasion and metastasis

doi: 10.1038/onc.2010.532

Figure Lengend Snippet: ( a ) HeyA8 cells (1×10 6 ) were injected intraperitoneally (i.p.) in nude mice which were randomized into 2 groups of 10/group receiving 10mg/kg human α 5 β 1 -integrin antibody (Hu α 5 Ab) or control IgG. The effect of Hu α 5 Ab on tumor weight was assessed on day 20. ( b ) SKOV3ip1 cells (1×10 6 ) were similarly injected i.p. treatment started on day 8 and tumor weight and the number of metastases assessed on day 35. Representative images of SKOV3ip1 tumors stained for Ki-67 (x400). Images of sections from 5 mice were quantified (**p<0.01). ( c ) SKOV3ip1 cells (1×10 6 ) were injected i.p. in nude mice randomized into 6 groups of 10 each and were treated with the indicated antibodies starting on day 8. Mice were euthanized on day 35 and the effect on tumor weight and number of metastases was assessed. (*p<0.001, **p<0.01,). ( d ) The effect of co-administering SKOV3ip1 cells with a single dose of Hu α 5 Ab injected i.p. in mice (prevention) was compared with treatment of SKOV3ip1 tumor bearing mice starting 8 days after injection of cancer cells (intervention). The effect of treatment on tumor weight and number of metastases was measured. ( e ) A single dose of murine α 5 Ab was co-injected with SKOV3ip1 cells i.p. in nude mice (prevention) and tumor weight and number of metastases was measured on day 35. ( f ) Kaplan-Meier curve for survival. SKOV3ip1 cells were injected i.p. and 10 mice/group were treated with the human α 5 Ab or control IgG twice a week starting from day 8 (p<0.0001; log-rank test).

Article Snippet: The function blocking antibodies against human α 5 β 1 -integrin (M200, referred to as Hu α 5 Ab), mouse α 5 β 1 -integrin (339.1, referred to as Murine α 5 Ab) and Avastin® (Genentech) were provided by Facet Biotech Corp (Redwood City, CA).

Techniques: Injection, Control, Staining

Journal: Oncogene

Article Title: Ligand independent activation of c-Met by fibronectin and α 5 β 1 -integrin regulates ovarian cancer invasion and metastasis

doi: 10.1038/onc.2010.532

Figure Lengend Snippet: ( a ) Left: HeyA8 cells were transfected with α 5 -integrin siRNA or treated with human α 5 Ab and plated on fibronectin followed by western blots for α 5 -Integrin, p-c-MetY 1230,1234,1235 and c-Met. Right: HeyA8 cells were plated on fibronectin, collagen or poly-L-Lysine and were treated with human α 5 Ab followed by immunoblotting for phospho-c-Met Y 1230,1234,1235 , c-Met and actin. ( b ) HeyA8 cells were plated on fibronectin or poly-L-Lysine and treated with human α 5 Ab for 24 h. Cell lysates were immunoprecipitated (IP) with an antibody against α 5 -integrin or c-Met, respectively followed by immunoblotting (WB) for α 5 -integrin or c-Met. ( c ) Confocal microscopy. HeyA8 cells were plated on fibronectin coated cover slips, detected with c-Met and α 5 -integrin antibodies followed by fluorescein or Alexa Fluor 594 labeled secondary antibodies respectively. ( d ) SKOV3ip1 xenografts were treated with Hu α 5 Ab or IgG as described in Figure 1 a . Left: Western blots for p-c-Met Y 1230,1234,1235 and c-Met using tumor lysates. Right: Image of tumor sections stained for p-c-Met Y 1200,1234,1235 (x200 and x400). Images of sections from 5 mice were quantified using Image J with color deconvolution (*p<0.001). ( e ) Human umbilical vein endothelial cells (HUVEC) were plated on fibronectin and treated with human α 5 Ab followed by western blots for p-c-MetY 1230,1234,1235 and c-Met.

Article Snippet: The function blocking antibodies against human α 5 β 1 -integrin (M200, referred to as Hu α 5 Ab), mouse α 5 β 1 -integrin (339.1, referred to as Murine α 5 Ab) and Avastin® (Genentech) were provided by Facet Biotech Corp (Redwood City, CA).

Techniques: Transfection, Western Blot, Immunoprecipitation, Confocal Microscopy, Labeling, Staining

Journal: Oncogene

Article Title: Ligand independent activation of c-Met by fibronectin and α 5 β 1 -integrin regulates ovarian cancer invasion and metastasis

doi: 10.1038/onc.2010.532

Figure Lengend Snippet: ( a ) Top: HeyA8 cells were transfected with α 5 -integrin siRNA or treated with human α 5 Ab and plated on fibronectin for 24h. Western blots were performed for p-FAK-Y 397 , p-FAK-Y 861 , FAK, and α 5 -integrin. ( b ) HeyA8 cells were transfected with CD2FAK (constitutively active FAK), CD2FAK-Y397F (mutated/inactive FAK) or CD2 (vector). Top : Expression and phosphorylation (Y 397 ) of CD2FAK (160kDa) was verified by western blotting. Bottom: Transfected HeyA8 cells were added to matrigel coated Boyden chamber with human α 5 Ab or IgG and allowed to invade for 24 h. The number of invaded cells was counted. ( c ) HeyA8 cells were plated on fibronectin and treated with human α 5 Ab or IgG for 24 h. Phosphorylated (p)-Src Y 416 and Src was detected by immunoblotting.

Article Snippet: The function blocking antibodies against human α 5 β 1 -integrin (M200, referred to as Hu α 5 Ab), mouse α 5 β 1 -integrin (339.1, referred to as Murine α 5 Ab) and Avastin® (Genentech) were provided by Facet Biotech Corp (Redwood City, CA).

Techniques: Transfection, Western Blot, Plasmid Preparation, Expressing, Phospho-proteomics

Journal: Oncogene

Article Title: Ligand independent activation of c-Met by fibronectin and α 5 β 1 -integrin regulates ovarian cancer invasion and metastasis

doi: 10.1038/onc.2010.532

Figure Lengend Snippet: Proposed role of ligand independent activation of c-Met involving a fibronectin/α 5 β 1 -integrin/c-Met/Src/FAK dependent signaling pathway.

Article Snippet: The function blocking antibodies against human α 5 β 1 -integrin (M200, referred to as Hu α 5 Ab), mouse α 5 β 1 -integrin (339.1, referred to as Murine α 5 Ab) and Avastin® (Genentech) were provided by Facet Biotech Corp (Redwood City, CA).

Techniques: Activation Assay