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1.
Fig. 3.

Fig. 3. From: The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor ?-Stimulated Aortic Endothelial Cells .

Plasmids used in the present study. A schematic diagram of the Fgd1 encoding vectors used in this study is shown.

Thomas Daubon, et al. Mol Cell Biol. 2011 November;31(22):4430-4441.
2.
Fig. 1.

Fig. 1. From: The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor ?-Stimulated Aortic Endothelial Cells .

Fgd family. Fgd1 is the founding member of a family of seven related Fgd1-like genes encoding modular proteins containing in sequence an N-terminal domain, a Dbl homology (DH) domain, an adjacent plekstrin homology (PH1) domain, a FYVE-finger domain (with the exception of FRG [Fgd1-related GEF]), and a second C-term PH domain (PH2). The N-terminal domain is highly variable and contains a cortactin/Mabp1 binding domain (CBD) within a proline-rich domain (PRD) in Fgd1, an actin-binding domain (FAB) in Fgd4 (Frabin) and a FERM domain in FRG. Numbers refer to amino acid residues.

Thomas Daubon, et al. Mol Cell Biol. 2011 November;31(22):4430-4441.
3.
Fig. 9.

Fig. 9. From: The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor ?-Stimulated Aortic Endothelial Cells .

Fgd1 regulates a novel noncanonical TGF-β pathway. Serum-starved BAE cells were stimulated with TGF-β for 30 min, and whole-cell lysates were examined for Smad1/5/8 and Smad2/3 phosphorylation by Western blotting. Similar Smad2/3 and Smad1 levels show no alteration in Smad expression. Depletion of Fgd1 was verified with Ab#1. Similar tubulin levels seen in all lanes demonstrate equal protein loading. (B) BAE cells were independently transfected with control siRNA or siRNAFgd1#2 and then left untreated or stimulated with TGF-β for 30 min. Representative confocal images of Smad4 and F-actin stainings are shown. (C) Smad4 nuclear signal was quantified on the basis of Hoechst staining using ImageJ software (50 cells for each condition) for each experiment (n = 3).

Thomas Daubon, et al. Mol Cell Biol. 2011 November;31(22):4430-4441.
4.
Fig. 7.

Fig. 7. From: The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor ?-Stimulated Aortic Endothelial Cells .

Constitutively active Fgd1 fails to induce podosomes rosettes. (A) Representative confocal images of BAE cells transfected with a plasmid encoding either GFP-Cdc42, myc-Fgd1-RKB3, showing the localization of the tagged protein at podosomes, or GFP-Fgd1-2DBDEL showing that a fraction of the protein localizes at the plasma membrane. The corresponding staining for F-actin highlights the subcortical skeleton. Bar, 5 μm. (B) Representative images showing individual staining for cortactin, vinculin, MT1-MMP, P-Src, and N-WASp in BAE cells transfected with GFP-V12Cdc42, GFP-Fgd1-RKB3, or GFP Fgd1-2DBDEL, analyzed together with F-actin. Merged images of each protein (green) with F-actin (red) show accumulation of the protein at the subcortical skeleton; the nuclei are highlighted with Hoechst (blue) (bar, 10 μm). (C) Representative images showing individual staining for cortactin or F-actin in BAE cells transfected with GFP-FL-Fgd1 or GFP-Fgd1Δ156-165, analyzed together with F-actin. Merged images of each protein (green) with F-actin (red) show colocalization of the proteins in podosomes rosettes (GFP-Fgd1) or loosely assembled dots (GFP-Fgd1Δ156-165). The nuclei are highlighted with Hoechst strain (blue) (bar, 10 μm).

Thomas Daubon, et al. Mol Cell Biol. 2011 November;31(22):4430-4441.
5.
Fig. 8.

Fig. 8. From: The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor ?-Stimulated Aortic Endothelial Cells .

Fgd1, but not Frabin, enhances BAE cell matrix-degrading activities. (A) Representative merged images of BAE cells transfected with GFP, GFP-FL-Fgd1, or GFP-Frabin encoding constructs, subsequently exposed to TGF-β for 24 h, stained for cortactin (white), F-actin (red), and nuclei (blue) are shown in the top panel (bar, 10 μm). Individual F-actin stainings in these images are shown in the bottom panel. (B) Same experiment as in panel A, performed with cells plated on a gelatin-FITC matrix and stained for cortactin and F-actin to record podosome-forming cells and assess matrix-degrading activities. The areas of gelatin degradation were quantified using ImageJ software. The total degradation area was then normalized for the number of cells for each experiment (n = 3) and is presented as a percentage of the control response measured in GFP-transfected cells arbitrarily taken as 100%.

Thomas Daubon, et al. Mol Cell Biol. 2011 November;31(22):4430-4441.
6.
Fig. 4.

Fig. 4. From: The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor ?-Stimulated Aortic Endothelial Cells .

Fgd1 is activated in response to TGF-β. (A) BAE cells stimulated with TGF-β for different time periods were subjected to a Cdc42G15A-GST pulldown assay, and the membranes were incubated with either anti-Fgd1 (Ab#1) or antiphosphotyrosine (4G10) antibodies. Similar Fgd1 and tubulin levels seen in all lanes of corresponding lysates demonstrate equal protein loading. (B) Fgd1 was knocked down in BAE cells, and a 30-min TGF-β stimulation was performed 24 h after the second siRNA transfection. A pulldown assay was then performed as in panel A. (C) BAE cells were stimulated with TGF-β for different time periods, and phosphoproteins were immunoprecipitated with 4G10 antibodies. Immunoprecipitated proteins were analyzed for Fgd1 content (Ab#1). Similar Fgd1 and tubulin levels seen in all lanes corresponding to cell lysates demonstrate equal protein loading. (D) BAE cells were transfected with GFP-FL-Fgd1 and, on the next day, stimulated with TGF-β for increased periods of time. GFP fusion proteins were immunoprecipitated with GFP antibodies and analyzed for phosphorylation (4G10) and Fgd1 content (Ab#1). Similar GFP-Fgd1 and tubulin levels seen in all lanes corresponding to cell lysates demonstrate equal protein loading. (E) BAE cells were treated for 1 h with 10 μM SU6656 and then stimulated with TGF-β for 30 min, and phosphoproteins were immunoprecipitated and analyzed as in panel C.

Thomas Daubon, et al. Mol Cell Biol. 2011 November;31(22):4430-4441.
7.
Fig. 2.

Fig. 2. From: The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor ?-Stimulated Aortic Endothelial Cells .

Fgd1 is expressed in aortic endothelial cells. (A) Cells were stimulated or not with 5 ng of TGF-β/ml for 30 min, lysed, and subjected to the pulldown assay using a Cdc42G15A-GST fusion protein. The samples and a fraction of the corresponding lysate were then analyzed by Western blotting with anti-Fgd1 (Ab#1), anti-αPIX, anti-βPIX, anti-Vav2, or anti-Zizimin1 antibodies. (B) Cells were independently transfected with different siRNAs (#1 to #3) targeting Fgd1 and then transfected with GFP or GFP-FL-Fgd1 encoding constructs. On the next day, cell lysates were prepared and analyzed by Western blotting, using either commercial anti-Fgd1 antibodies (Ab#1) or homemade anti-Fgd1 antibodies (Ab#2). (C) Fgd1 protein expression was inhibited using siRNA Fgd1 #2, and TGF-β stimulation was performed 24 h after the second siRNA transfection. Immunostainings for Fgd1 were performed with anti-Fgd1 antibody (Ab#1 or Ab#2) revealed with Alexa 488 secondary antibodies, together with phalloidin-Alexa 546 and Hoechst. Bar, 10 μm.

Thomas Daubon, et al. Mol Cell Biol. 2011 November;31(22):4430-4441.
8.
Fig. 6.

Fig. 6. From: The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor ?-Stimulated Aortic Endothelial Cells .

Fgd1 and cortactin relocalize at the plasma membrane upon TGF-β stimulation. (A) Cells were treated with TGF-β for increasing time periods, fixed, and analyzed by immunofluorescence for Fgd1 (Ab#2), Cdc42-GTP, cortactin, and F-actin. Representative confocal images show individual staining for Fgd1, Cdc42-GTP, cortactin, and F-actin (bar, 10 μm). A magnified view of the boxed zone in the F-actin image shows the merge image of Fgd1 (green) and Cdc42 (red) or Fgd1 (green) and cortactin (red) stainings and their colocalization (yellow). (B) Western blot analysis of BAE cells extracts after cell fractionation, showing cytosolic and membrane/cytoskeleton-bound Fgd1 or cortactin in the course of TGF-β stimulation without serum. The purity of the extracts was assessed by reprobing the membrane for tubulin (cytoplasmic extracts) and CD31 (membrane extracts). (C) BAE cells were stimulated with TGF-β for increasing periods of time, and cortactin was immunoprecipitated. Samples were analyzed by Western blotting for cortactin and Fgd1 content (Ab#1). Similar Fgd1 and cortactin levels seen in all lanes corresponding to cell lysates demonstrate equal protein loading. (D) Same experiments as in panel A performed with cells which had been depleted of Fgd1 or cortactin by transfection of siRNA Fgd1 #2 or cortactin siRNA and subsequently stimulated with TGF-β for 30 min (bar, 10 μm). A magnified view of the boxed zone in the merge image shows details of Fgd1 and cortactin stainings (yellow) (bar, 10 μm). (E) Western blot analysis showing Fgd1 and cortactin expression in whole-cell lysates prepared from BAE cell cultures used in experiment depicted in panel D showing effective depletion of the targeted proteins.

Thomas Daubon, et al. Mol Cell Biol. 2011 November;31(22):4430-4441.
9.
Fig. 5.

Fig. 5. From: The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor ?-Stimulated Aortic Endothelial Cells .

Depletion of Fgd1 impairs TGF-β-induced podosome formation and Cdc42 activation. (A) Fgd1 was silenced in BAE cells using either of the three described siRNAs, and TGF-β stimulation was performed 24 h after the second siRNA transfection. For each condition, cells showing podosome rosettes were recorded and compared to the value obtained in the control siRNA condition, which was fixed arbitrarily at 100% for each experiment (n = 4). Alternatively, transfected cells were seeded on a gelatin-FITC matrix and stimulated with TGF-β for 24 h. Cells were fixed and double stained for F-actin and cortactin to record podosome-forming cells and assess matrix-degrading activities. The areas of gelatin degradation were quantified using ImageJ software, and the total degradation area was then normalized for the number of cells for each experiment (n = 3). (B) Western blot analysis of Fgd1 depletion in cells subjected to siRNA transfection used for the experiment described above. Similar tubulin levels seen in all lanes demonstrate equal protein loading. (C) BAE cells silenced for Fgd1 expression using siRNA#2 were transfected with a plasmid encoding GFP or GFP-FL-Fgd1 and stimulated for 24 h with TGF-β on the next day. (D) Western blot analysis of Fgd1 depletion and of Fgd1 reexpression using anti-GFP and anti-Fgd1 (Ab#1) antibodies in siRNA#2 silenced cells transfected with constructs encoding GFP-FL-Fgd1 or GFP alone in panel C showing effective depletion of the targeted proteins. (E) Quantification of cells showing podosome rosettes was performed in Fgd1 silenced BAE cells transfected with constructs encoding GFP-FL-Fgd1 or GFP alone, using GFP fluorescence. (F) The alterations of Cdc42 activities in BAE cells exposed to TGF-β for 4 or 24 h was assessed in pulldown assays using GST-CRIB-N-WASp fusion proteins, and the amounts of precipitated proteins were determined by Western blotting with specific antibodies. A fraction of whole-cell lysate was harvested and run in parallel to assess total Cdc42 protein contents. Similar tubulin levels in all lanes demonstrate equal protein loading. (G) Quantification of Cdc42 activation after scanning band intensities in the autoradiogram and processing of the data with NIH Image software (n = 3).

Thomas Daubon, et al. Mol Cell Biol. 2011 November;31(22):4430-4441.

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