Figure 1.
Ligand-independent role of ErbB3 in epithelial sheet motility. (A) Protein expression of ErbB3 and GAPDH (as a loading control) in the cell extract of MCF10A transiently transfected with control siRNA (siCtrl) or siRNA targeting ErbB3 (#2). Densitometric values of ErbB3 protein expression in three biological replicates ± SEM of MCF10A cells are shown normalized to the respective siCtrl. Data are presented as mean values ± SEM. P values were determined by unpaired Student’s t test. **P ≤ 0.01. (B) Scratch closure assay of MCF10A cells transiently transfected with siCtrl or siErbB3#2, cultured in serum-containing but growth factor–deprived media in the presence of DMSO (vehicle) or 1 µM of the EGFR/ErbB2 inhibitor lapatinib. The wound area is highlighted in yellow. (C and D) Quantification of the scratch aperture (C) or AUC (D) of samples treated as in B. Data are presented as mean values ± SEM; n values are indicated in the parenthesis. (E) Quantification of cell proliferation as incorporation of EdU for the indicated times in control or ErbB3 siRNA#2-transfected cells in the presence of DMSO (vehicle) or 1 µM lapatinib. Data are presented as mean values ± SEM; n = 3 independent experiments. (F–I) Confocal immunofluorescence imaging of surface-labeled Integrin β1 (green) or Actin (blue/black) on confluent sheets of MCF10A cells transfected with control siRNA (siCtrl) or siErbB3#2 at 0 or 1 h after labeling (G), as outlined in F. The boxed regions in G indicate the leading edge of the wound within a closing cell sheet. (H) Integrin β1 enrichment was determined as ((a−b)/b), where a = mean fluorescence intensity (Integrin β1) at a defined area of the leading edge (H) or cell–cell contact (I) and b = mean intensity of adjacent cytoplasm of the same area. Data are presented as mean values (>74 cells per data point) ± SEM; n = 3 independent experiments. Scale bars, 1 mm (B) and 10 µm (G).

Ligand-independent role of ErbB3 in epithelial sheet motility. (A) Protein expression of ErbB3 and GAPDH (as a loading control) in the cell extract of MCF10A transiently transfected with control siRNA (siCtrl) or siRNA targeting ErbB3 (#2). Densitometric values of ErbB3 protein expression in three biological replicates ± SEM of MCF10A cells are shown normalized to the respective siCtrl. Data are presented as mean values ± SEM. P values were determined by unpaired Student’s t test. **P ≤ 0.01. (B) Scratch closure assay of MCF10A cells transiently transfected with siCtrl or siErbB3#2, cultured in serum-containing but growth factor–deprived media in the presence of DMSO (vehicle) or 1 µM of the EGFR/ErbB2 inhibitor lapatinib. The wound area is highlighted in yellow. (C and D) Quantification of the scratch aperture (C) or AUC (D) of samples treated as in B. Data are presented as mean values ± SEM; n values are indicated in the parenthesis. (E) Quantification of cell proliferation as incorporation of EdU for the indicated times in control or ErbB3 siRNA#2-transfected cells in the presence of DMSO (vehicle) or 1 µM lapatinib. Data are presented as mean values ± SEM; n = 3 independent experiments. (F–I) Confocal immunofluorescence imaging of surface-labeled Integrin β1 (green) or Actin (blue/black) on confluent sheets of MCF10A cells transfected with control siRNA (siCtrl) or siErbB3#2 at 0 or 1 h after labeling (G), as outlined in F. The boxed regions in G indicate the leading edge of the wound within a closing cell sheet. (H) Integrin β1 enrichment was determined as ((a−b)/b), where a = mean fluorescence intensity (Integrin β1) at a defined area of the leading edge (H) or cell–cell contact (I) and b = mean intensity of adjacent cytoplasm of the same area. Data are presented as mean values (>74 cells per data point) ± SEM; n = 3 independent experiments. Scale bars, 1 mm (B) and 10 µm (G).

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