Figure S1.
ErbB3 promotes Integrin β1 endocytic recycling. (A) Viability curves of MCF7 and MCF10A cells in the presence of increasing concentrations of lapatinib (logarithmic scale) assessed by PrestoBlue fluorescence. Note the lack of significant differences. (B) Protein expression levels of the indicated signaling proteins and GAPDH (as a loading control) in MCF10A cells stimulated with 20 ng/ml EGF in the absence or presence of 1 µM lapatinib for 30 min. Average densitometric values of at least two independent biological replicates were normalized to the respective control condition (EGF-negative; lapatinib-positive). (C) RT-qPCR analysis of the indicated mRNA levels in MCF7, MCF10A, and prHMEC cells. Values represent fold change of ErbB3 (left) and Integrin β1 (ITGB1; right) mRNA expression relative to GAPDH. Data are presented as mean values of three biological replicates ± SEM, each in technical duplicates. (D) Analysis of colocalization of endogenous traced Integrin β1, ErbB3, and the recycling endosomal marker EDH1: MCF7 cells were labeled on ice with an Alexa 488–conjugated anti-Integrin β1 antibody (green) prior to incubation for 30 min at 37°C to allow Integrin β1 internalization, and subsequent immunolabeling of ErbB3 (magenta), counterstained for EDH1 (white, right panel). Note that the squared region in the left panel represents the magnified images of D. (E and G) Protein expression of ErbB3 and GAPDH (as a loading control) in the cell extract of MCF10A (E) and prHMEC (G) cells transiently transfected with control siRNA (siCtrl) or two independent siRNAs targeting ErbB3 (#1 and #2). Densitometric values of ErbB3 protein expression in three biological replicates ± SEM of MCF10A cells are shown normalized to the respective siCtrl. (F and H) Columns show the AUC of the Integrin β1 recycling data in MCF10A (F: related to Fig. 2 G) and prHMEC cells (H: related to Fig. 2 I). Data are presented as mean values ± SEM; n values are indicated in the main figures. (I–L) Immunofluorescence imaging of the surface pool of Integrin β1 labeled with Alexa 488–conjugated antibody on ice for 1 h, on MCF10A (I and J) or prHMEC cells (K and L) transfected with siCtrl or ErbB3-targeting siRNAs. The columns in J and L show quantification of Integrin β1 fluorescence intensity, normalized against control siRNA-treated samples. Data are presented as the mean ± SEM, from six independent experiments. P values in C, E, F, H, J, and L were determined by one-way ANOVA followed by multiple paired comparisons conducted by Bonferroni’s posttest method. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, nonsignificant. Scale bars, 10 µm (D), 20 µm (I and K).

ErbB3 promotes Integrin β1 endocytic recycling. (A) Viability curves of MCF7 and MCF10A cells in the presence of increasing concentrations of lapatinib (logarithmic scale) assessed by PrestoBlue fluorescence. Note the lack of significant differences. (B) Protein expression levels of the indicated signaling proteins and GAPDH (as a loading control) in MCF10A cells stimulated with 20 ng/ml EGF in the absence or presence of 1 µM lapatinib for 30 min. Average densitometric values of at least two independent biological replicates were normalized to the respective control condition (EGF-negative; lapatinib-positive). (C) RT-qPCR analysis of the indicated mRNA levels in MCF7, MCF10A, and prHMEC cells. Values represent fold change of ErbB3 (left) and Integrin β1 (ITGB1; right) mRNA expression relative to GAPDH. Data are presented as mean values of three biological replicates ± SEM, each in technical duplicates. (D) Analysis of colocalization of endogenous traced Integrin β1, ErbB3, and the recycling endosomal marker EDH1: MCF7 cells were labeled on ice with an Alexa 488–conjugated anti-Integrin β1 antibody (green) prior to incubation for 30 min at 37°C to allow Integrin β1 internalization, and subsequent immunolabeling of ErbB3 (magenta), counterstained for EDH1 (white, right panel). Note that the squared region in the left panel represents the magnified images of D. (E and G) Protein expression of ErbB3 and GAPDH (as a loading control) in the cell extract of MCF10A (E) and prHMEC (G) cells transiently transfected with control siRNA (siCtrl) or two independent siRNAs targeting ErbB3 (#1 and #2). Densitometric values of ErbB3 protein expression in three biological replicates ± SEM of MCF10A cells are shown normalized to the respective siCtrl. (F and H) Columns show the AUC of the Integrin β1 recycling data in MCF10A (F: related to Fig. 2 G) and prHMEC cells (H: related to Fig. 2 I). Data are presented as mean values ± SEM; n values are indicated in the main figures. (I–L) Immunofluorescence imaging of the surface pool of Integrin β1 labeled with Alexa 488–conjugated antibody on ice for 1 h, on MCF10A (I and J) or prHMEC cells (K and L) transfected with siCtrl or ErbB3-targeting siRNAs. The columns in J and L show quantification of Integrin β1 fluorescence intensity, normalized against control siRNA-treated samples. Data are presented as the mean ± SEM, from six independent experiments. P values in C, E, F, H, J, and L were determined by one-way ANOVA followed by multiple paired comparisons conducted by Bonferroni’s posttest method. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, nonsignificant. Scale bars, 10 µm (D), 20 µm (I and K).

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