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1.
FIG. 3.

FIG. 3. From: Signaling through ShcA Is Required for Transforming Growth Factor ?- and Neu/ErbB-2-Induced Breast Cancer Cell Motility and Invasion .

Signaling downstream of tyrosine residues 1226/1227 (YD) and 1253 (YE) in Neu/ErbB-2 synergize with TGF-β to promote cell motility and invasion. Explant cultures from mammary tumors expressing Neu/ErbB-2 add-back mutants were subjected to motility (A) and invasion (B) assays as described in Materials and Methods. Representative images from explant cultures expressing each Neu/ErbB-2 add-back receptor are shown (right) for both motility (A) and invasion (B). Significant differences in cell motility (A) (*, P < 0.02; **, P < 0.04) and invasion (B) (*, P < 0.02; **, P < 0.03) were observed in Neu-YD and Neu-YE tumor explants following TGF-β stimulation.

Jason J. Northey, et al. Mol Cell Biol. 2008 May;28(10):3162-3176.
2.
FIG. 6.

FIG. 6. From: Signaling through ShcA Is Required for Transforming Growth Factor ?- and Neu/ErbB-2-Induced Breast Cancer Cell Motility and Invasion .

Transient siRNA-mediated knockdown of the ShcA adaptor protein in Neu/ErbB-2-expressing breast cancer explants. (A) Immunoblot analysis of Neu-NT-expressing NMuMG breast cancer cells treated with a scrambled control siRNA (C) or a mixture of three siRNAs targeting ShcA (S). Lysates were prepared 1, 2, 3, and 4 days following the transfection (Post-transfect.) of the siRNAs. (B) The effectiveness of the ShcA knockdowns was confirmed 3 days following transfection in Neu-NT-, Neu-YC-, Neu-YD-, and Neu-YE-expressing NMuMG breast cancer cells.

Jason J. Northey, et al. Mol Cell Biol. 2008 May;28(10):3162-3176.
3.
FIG. 4.

FIG. 4. From: Signaling through ShcA Is Required for Transforming Growth Factor ?- and Neu/ErbB-2-Induced Breast Cancer Cell Motility and Invasion .

Neu-NT-expressing mammary tumor explants form fewer and smaller focal adhesions in response to TGF-β treatment than empty vector- or Neu-NYPD-expressing explants. Immunofluorescence staining was performed for FAK (red) and actin (green) on all three explant populations in the absence or presence of TGF-β (24 h). DAPI staining (blue) was performed to visualize nuclei. The number of focal adhesions formed under each condition was quantified using Volocity software. Quantified data are expressed as the number of focal adhesions per DAPI-stained nucleus and were obtained from five fields for each condition from three independent experiments. The scale bar represents 10 μm and applies to all panels.

Jason J. Northey, et al. Mol Cell Biol. 2008 May;28(10):3162-3176.
4.
FIG. 7.

FIG. 7. From: Signaling through ShcA Is Required for Transforming Growth Factor ?- and Neu/ErbB-2-Induced Breast Cancer Cell Motility and Invasion .

Loss of ShcA expression renders Neu-NT-, Neu-YD-, and Neu-YE-expressing NMuMG breast cancer cells refractory to TGF-β-induced motility and invasion. Explant cultures from mammary tumors expressing Neu-NT, Neu-YC, Neu-YD, and Neu-YE were subjected to motility (A) and invasion (B) assays as described in Materials and Methods. For each explant, cells were transfected with a scrambled control siRNA (C) or a pool of siRNAs targeting ShcA (S) prior to a 24-h incubation in the absence or presence of TGF-β. Representative images from control and ShcA siRNA-treated explant cultures, in the absence or presence of TGF-β, are shown (lower) for both motility (A) and invasion (B). Significant differences in cell motility (A) (*, P < 0.00097; **, P < 0.00005; ***, P < 0.01134) and invasion (B) (*, P < 0.02673; **, P < 0.00256; ***, P < 0.00946) were observed in Neu-NT, Neu-YD, and Neu-YE tumor explants following TGF-β stimulation.

Jason J. Northey, et al. Mol Cell Biol. 2008 May;28(10):3162-3176.
5.
FIG. 5.

FIG. 5. From: Signaling through ShcA Is Required for Transforming Growth Factor ?- and Neu/ErbB-2-Induced Breast Cancer Cell Motility and Invasion .

Neu-YD-expressing explants display the lowest level of density of mature focal adhesions, which is associated with enhanced cell motility and invasion, in response to TGF-β stimulation. Immunofluorescence staining was performed for FAK (red) and actin (green) on all Neu add-back populations (Neu-YA, Neu-YB, Neu-YC, Neu-YD, and Neu-YE) in the absence or presence of TGF-β (24 h). DAPI staining (blue) was performed to visualize nuclei. The number of focal adhesions formed under each condition was quantified using Volocity software. Quantified data are expressed as the number of focal adhesions per DAPI-stained nucleus and were obtained from five fields for each condition from three independent experiments. The scale bar represents 10 μm and applies to all panels.

Jason J. Northey, et al. Mol Cell Biol. 2008 May;28(10):3162-3176.
6.
FIG. 8.

FIG. 8. From: Signaling through ShcA Is Required for Transforming Growth Factor ?- and Neu/ErbB-2-Induced Breast Cancer Cell Motility and Invasion .

Reduced ShcA expression results in the formation of increased numbers of large focal adhesions in Neu-NT-, Neu-YD-, and Neu-YE-expressing mammary tumor cells following TGF-β stimulation. Immunofluorescence staining was performed for FAK (A) and vinculin (B) on Neu-NT-, Neu-YC-, Neu-YD-, and Neu-YE-expressing cells that were treated with a scrambled control siRNA (C) or a cocktail of three siRNAs targeting ShcA. The cells were incubated in the absence (−) or presence (+) of TGF-β for 24 h. The number of FAK-stained (A) or vinculin-stained (B) focal adhesions formed under each condition was quantified using Volocity software. Quantified data are expressed as the number of focal adhesions per DAPI-stained nuclei and were obtained from five fields for each condition from three independent experiments.

Jason J. Northey, et al. Mol Cell Biol. 2008 May;28(10):3162-3176.
7.
FIG. 2.

FIG. 2. From: Signaling through ShcA Is Required for Transforming Growth Factor ?- and Neu/ErbB-2-Induced Breast Cancer Cell Motility and Invasion .

TGF-β stimulates the motility and invasion of Neu-NT-expressing mammary tumor explants. A single explant population of control NMuMG cells harboring the empty vector (Empty Explant 117R) and two tumor explants from Neu-NT-expressing (0118R and 0118L) and Neu-NYPD-expressing (0120R and 0121L) tumors were left untreated or were treated with TGF-β for 24 h, harvested, and plated for motility (A) and invasion assays (B) as described in Materials and Methods. The data represent results from three independent experiments for each cell population, with duplicate wells counted for each condition. Five images from each filter were quantified using Scion Image software. Representative images from one set of explant populations harboring each construct are shown (right) for both motility (A) and invasion (B). Significant differences in cell motility (A) (*, P < 0.05; **, P < 0.03) and invasion (B) (*, P < 0.002; **, P < 0.01) were observed only in TGF-β-treated Neu-NT-expressing tumor explants.

Jason J. Northey, et al. Mol Cell Biol. 2008 May;28(10):3162-3176.
8.
FIG. 1.

FIG. 1. From: Signaling through ShcA Is Required for Transforming Growth Factor ?- and Neu/ErbB-2-Induced Breast Cancer Cell Motility and Invasion .

Neu/ErbB-2-transformed NMuMG cells form mammary tumors in vivo. (A) Immunoblot analysis on pooled stable populations of NMuMG cells expressing either Neu-NT, Neu-NYPD, or the empty vector control using antibodies against Neu/ErbB-2 and α-tubulin. (B) Mammary tumor outgrowth was measured by weekly caliper measurements following the injection of pooled stables into the fourth mammary fat pad. The average tumor volumes (± standard deviations) from four independent tumors expressing Neu-NT or Neu-NYPD are plotted (*, P < 0.0013), whereas NMuMG empty vector control cells did not form palpable tumors following injection. (C) Immunoblot analyses were performed on whole-cell lysates from the indicated explant cultures with both Neu/ErbB-2- and α-tubulin-specific antibodies (left). Neu/ErbB-2 immunoprecipitations also were performed, and the immunoprecipitates were split and blotted for phosphotyrosine and Neu/ErbB-2 (right). (D) Explant cultures were reinjected into the fourth mammary fat pad, and mammary tumor outgrowth was measured by weekly caliper measurement (**, P < 0.0001). (E) Neu/ErbB-2- and α-tubulin-specific immunoblot analyses were performed on protein lysates prepared from Neu-NT- and Neu-NYPD-expressing mammary tumors (numbers refer to mouse ear tag numbers) (left). Neu/ErbB-2 was immunoprecipitated from Neu-NT- and Neu-NYPD-expressing mammary tumor lysates in duplicate; one set of immunoprecipitates was blotted for phosphotyrosine, and the other was blotted for Neu/ErbB-2 (right). IP, immunoprecipitation; P-Tyr, phosphotyrosine.

Jason J. Northey, et al. Mol Cell Biol. 2008 May;28(10):3162-3176.

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