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. Author manuscript; available in PMC: 2016 Mar 1.
Published in final edited form as: Mol Cancer Res. 2014 Oct 15;13(3):423–438. doi: 10.1158/1541-7786.MCR-14-0353

Figure 7. Depletion of CAV1 rescues integrin trafficking, adhesion and migration of NEDD9 deficient cells.

Figure 7

(A) WB analysis of MDA-MB-231-siCon; -siN2, -siN3 cells treated or untreated with siCAV1 (+/−) and expressing either pGFP empty vector (EV-Panel 1), or pGFP-Cav1-wt or -Y14F (Panel 2); staining with anti-CAV1 (labeled as Cav-1 and GFP-Cav1), -NEDD9, -tubulin antibodies. (B) FACS of ligand-bound β1 integrin cells as in (A) Panel 1; RFU, (%) to siCon + SEM; n=3; one-way ANOVA *p<0.002 (siCon+EV/siN2–3+EV,); p=ns (siCon+EV/siCAV1+EV+siN2–3). (C) Quantification of volume of early endosomes in the same cells as in (A) based on 3D reconstructed confocal images with anti-EEA1; mean volume, (%) to control + SEM; n=3; 20 cells/treatment; one-way ANOVA: *p<0.01 (siCon+EV/siN2–3+EV); p<0.0001 (siN2–3+EV/siN2–3+siCAV1+EV); p<0.02 (siN2–3+siCAV1+EV/siN2–3+siCAV1+Cav1-wt); p=ns (siN2–3+siCAV1+EV/siN2–3+siCAV1+Cav1-Y14F). (D) Boyden chamber migration assay. Quantification of RFU % to siCon+SEM; n=3; cells as in (A); one-way ANOVA: *p<0.003 (siCon+EV/siN2–3+EV); p<0.0001 (siN2–3+EV/siN2–3+siCAV1+EV); p=ns (siN2–3+EV/siN2–3+siCAV1+Cav1-wt); p<0.006 (siN2–3+EV/siN2–3+siCAV+Cav1-Y14F); p=ns (siN2–3+siCAV1+EV/siN2–3+siCAV1+Cav1-Y14F). (E) Adhesion and spreading assay; cells as in (A). Representative bright field images after 1 h of adhesion/spreading; dotted lines represent cell borders; scale bar, 10µm; (F) Adhesion and spreading assay. Quantification of average area of cell as in (E) % to siCon + SEM; n=3; one-way ANOVA *p<0.0001 (siCon+EV/siN2–3+EV); p<0.0001 (siN2–3+EV/siN2–3+siCAV1+EV); p=ns (siN2–3+EV/siN2–3+siCAV1+Cav1-wt); p<0.0001 (siN2–3+EV/siN2–3+siCAV+Cav1-Y14F), p=ns (siN2–3+siCAV1+EV/siN2–3+siCAV1+Cav1-Y14F). (G) The schematic model: The internalized through caveolae integrins delivered to early sorting endosomes; ligand-free integrins will be targeted back to plasma membrane through the fast recycling route, while ligand-bound integrins will be trafficked to late endosomes/lysosomes through the slow recycling route. NEDD9-dependent dephosphorylation of pTyr14-Cav1 is required for sorting ligand-bound integrins to late endosomes to enable disengage of integrins from ligand.