Figure 2.
ErbB3 promotes Integrin β1 andTfR endocytic recycling. (A–D) Confocal immunofluorescence imaging of traced surface-labeled Integrin β1 and ErbB3: MCF7 cells were labeled on ice with an Alexa 488–conjugated anti-Integrin β1 antibody prior to incubation for 15 min at 37°C to allow Integrin β1 internalization, and subsequent cell fixation and immunolabeling of ErbB3 (red) and counterstaining with DAPI (blue). Note that B represents the magnified (squared) regions of A. (C) Histogram of fluorescence intensities along dotted lines indicated in B. (D) Analysis of Integrin β1 and ErbB3 colocalization. Integrin β1 enrichment in ErbB3-positive intracellular structures (0.5–2 µm diameter) was determined by the formula (a−b)/b, where a is the Integrin β1 intensity at ErbB3-positive structures, and b is the adjacent intensity (background) for each structure. Average intensity projections of all analyzed structures are shown on the right-hand side. (E) Schematic outline of Integrin β1 recycling assays conducted in F–K after transfection with siRNA against the indicated targets. Integrin β1 surface pool was labeled with an Alexa 488–conjugated antibody and allowed to endocytose. Fluorophore label remaining on the cell surface was quenched with an anti-Alexa 488 antibody, prior to visualization of traced Integrin β1 reemerging on the cell surface by live-cell TIRF microscopy. (F) Representative TIRF microscopy images of Integrin β1 from peripheral areas of MCF10A cells transiently transfected with control siRNA (siCtrl), siErbB3#1, or siErbB3#2. (G) Quantifications of recycled Integrin β1 performed on indicated number of cells (outside of brackets on the right-hand side of graphs), from three independent experiments and shown as Alexa 488 intensity normalized between 0 and 1, with the control as reference where Fnorm=((Fmax-Fmin)/(F-Fmin)). (H) Representative TIRF microscopy images of Integrin β1 from prHMEC cells transiently transfected with siCtrl, siErbB3#1, or siErbB3#2. (I) Quantifications of recycled Integrin β1 in prHMEC performed as described in E. (J and K) Quantified Integrin β1 recycling, after siRNA-mediated depletion of either EGFR (J) or ErbB2 (K). Data are presented as mean values ± SEM, and P values were determined by two-tailed paired Student’s t test. ns, nonsignificant. (L) Schematic outline of transferrin recycling assays. (M) Confocal imaging of Alexa 594–conjugated transferrin chased with unlabeled holo-transferrin for the indicated times in MCF7 cells. (N) Quantification of Alexa 594 fluorescence intensity in cells treated as in M (n > 17 cells for each data point from three experiments) normalized against the control siRNA-treated samples, 0-h time point of each independent experiment. Scale bars, 7 µm (A), 4 µm (B), 10 µm (F, H, and M), and 0.5 µm (D).

ErbB3 promotes Integrin β1 and TfR endocytic recycling. (A–D) Confocal immunofluorescence imaging of traced surface-labeled Integrin β1 and ErbB3: MCF7 cells were labeled on ice with an Alexa 488–conjugated anti-Integrin β1 antibody prior to incubation for 15 min at 37°C to allow Integrin β1 internalization, and subsequent cell fixation and immunolabeling of ErbB3 (red) and counterstaining with DAPI (blue). Note that B represents the magnified (squared) regions of A. (C) Histogram of fluorescence intensities along dotted lines indicated in B. (D) Analysis of Integrin β1 and ErbB3 colocalization. Integrin β1 enrichment in ErbB3-positive intracellular structures (0.5–2 µm diameter) was determined by the formula (a−b)/b, where a is the Integrin β1 intensity at ErbB3-positive structures, and b is the adjacent intensity (background) for each structure. Average intensity projections of all analyzed structures are shown on the right-hand side. (E) Schematic outline of Integrin β1 recycling assays conducted in F–K after transfection with siRNA against the indicated targets. Integrin β1 surface pool was labeled with an Alexa 488–conjugated antibody and allowed to endocytose. Fluorophore label remaining on the cell surface was quenched with an anti-Alexa 488 antibody, prior to visualization of traced Integrin β1 reemerging on the cell surface by live-cell TIRF microscopy. (F) Representative TIRF microscopy images of Integrin β1 from peripheral areas of MCF10A cells transiently transfected with control siRNA (siCtrl), siErbB3#1, or siErbB3#2. (G) Quantifications of recycled Integrin β1 performed on indicated number of cells (outside of brackets on the right-hand side of graphs), from three independent experiments and shown as Alexa 488 intensity normalized between 0 and 1, with the control as reference where Fnorm=((Fmax-Fmin)/(F-Fmin)). (H) Representative TIRF microscopy images of Integrin β1 from prHMEC cells transiently transfected with siCtrl, siErbB3#1, or siErbB3#2. (I) Quantifications of recycled Integrin β1 in prHMEC performed as described in E. (J and K) Quantified Integrin β1 recycling, after siRNA-mediated depletion of either EGFR (J) or ErbB2 (K). Data are presented as mean values ± SEM, and P values were determined by two-tailed paired Student’s t test. ns, nonsignificant. (L) Schematic outline of transferrin recycling assays. (M) Confocal imaging of Alexa 594–conjugated transferrin chased with unlabeled holo-transferrin for the indicated times in MCF7 cells. (N) Quantification of Alexa 594 fluorescence intensity in cells treated as in M (n > 17 cells for each data point from three experiments) normalized against the control siRNA-treated samples, 0-h time point of each independent experiment. Scale bars, 7 µm (A), 4 µm (B), 10 µm (F, H, and M), and 0.5 µm (D).

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