Figure 4.
ErbB3 loss directs Integrin β1 toward lysosomal degradation or EV secretion. (A) Schematic outline of traced internalized integrin. (B) Confocal immunofluorescence imaging of traced internalized Integrin β1 in MCF10A cells transiently transfected with control siRNA (siCtrl) or siErbB3#2. (C) Quantification of immunofluorescence intensity of internalized Integrin β1 that was traced for the indicated times after a 15-min internalization step (n > 32 cells per data point from 5 independent experiments). (D) Determination of Integrin β1 turnover by pulse-chase metabolic labeling: control or ErbB3 siRNA#1-transfected MCF10A cells were pulse chase–labeled with radioactive (35S) methionine and cysteine. Radiolabeled Integrin β1 was visualized by radiography of immunoprecipitates (upper panel). Cell lysates and immunoprecipitates were analyzed by immunoblotting. (E) Quantification of pulse-chased 35S-labeled Integrin β1 (left panel, siErbB3#1; and right panel, siErbB3#2) (n = 4 independent experiments). (F and G) Confocal immunofluorescence imaging of surface-labeled Integrin β1 (using an Alexa 488–conjugated anti-Integrin β1 antibody), prior to (0 h) or after tracing at 37°C for 1.5 h. A scratch was inflicted prior to antibody incubation. Note that application of the lysosome inhibitor chloroquine caused Integrin β1 accumulation in intracellular vesicular compartments in MCF10A transiently transfected with siCtrl or siErbB3#2. (H) Quantification of Integrin β1 fluorescence intensity in cells bordering the migratory front in samples treated as in G, showing that chloroquine restored Integrin β1 levels in ErbB3-depleted cells. Data were presented as mean values ± SEM; n = 19–27 cells per data point from 3 independent experiments. P values were determined by two-tailed paired Student’s t test. ns, nonsignificant. (I) Protein expression of Integrin β1 and the indicated EV markers in the UC EV protein extract; average densitometric values of at least three independent biological replicates were normalized to siCtrl. (J) Representative TEM micrographs of EVs enriched by ultracentrifugation (UC EVs) from control or ErbB3-depleted MCF10A cells and stained with gold-conjugated anti-CD63 antibody (CD63-EVs are indicated by red arrows). (K) Cell viability assay measured with PrestoBlue Cell Viability Reagent in MCF10A cells transiently transfected with siCtrl or siErbB3#2 incubated with EV-free media as vehicle (Ctrl) or 1 × 109 nanoparticles of EVs secreted from MCF10A cells transiently transfected with control (VSFsiCtrl) or ErbB3-siRNA (VSFsiErbB3) for 48 h. (L) Matrigel invasion assay in transwells with MCF10A cells stimulated with EV-free media (Ctrl) or incubated with equal number of VSFsiCtrl or VSFsiErbB3 as described in K for 16 h. (M) Matrigel invasion assay in transwells with MCF10A WT cells prestimulated for 48 h with EV-free media (Ctrl) or incubated with equal number of VSFsiCtrl or VSFsiErbB3 in the presence of 5 ug/ml IgG or Integrin β1 monoclonal antibody (mAb; P4C10) for an additional 16 h. The data in K–M are presented as mean values of three biological replicates ±SEM and P values shown based on two-way ANOVA, followed by multiple paired comparisons conducted by means of Bonferroni’s posttest method (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). Scale bars, 15 µm (A), 10 µm (G), 200 nm (J).

ErbB3 loss directs Integrin β1 toward lysosomal degradation or EV secretion. (A) Schematic outline of traced internalized integrin. (B) Confocal immunofluorescence imaging of traced internalized Integrin β1 in MCF10A cells transiently transfected with control siRNA (siCtrl) or siErbB3#2. (C) Quantification of immunofluorescence intensity of internalized Integrin β1 that was traced for the indicated times after a 15-min internalization step (n > 32 cells per data point from 5 independent experiments). (D) Determination of Integrin β1 turnover by pulse-chase metabolic labeling: control or ErbB3 siRNA#1-transfected MCF10A cells were pulse chase–labeled with radioactive (35S) methionine and cysteine. Radiolabeled Integrin β1 was visualized by radiography of immunoprecipitates (upper panel). Cell lysates and immunoprecipitates were analyzed by immunoblotting. (E) Quantification of pulse-chased 35S-labeled Integrin β1 (left panel, siErbB3#1; and right panel, siErbB3#2) (n = 4 independent experiments). (F and G) Confocal immunofluorescence imaging of surface-labeled Integrin β1 (using an Alexa 488–conjugated anti-Integrin β1 antibody), prior to (0 h) or after tracing at 37°C for 1.5 h. A scratch was inflicted prior to antibody incubation. Note that application of the lysosome inhibitor chloroquine caused Integrin β1 accumulation in intracellular vesicular compartments in MCF10A transiently transfected with siCtrl or siErbB3#2. (H) Quantification of Integrin β1 fluorescence intensity in cells bordering the migratory front in samples treated as in G, showing that chloroquine restored Integrin β1 levels in ErbB3-depleted cells. Data were presented as mean values ± SEM; n = 19–27 cells per data point from 3 independent experiments. P values were determined by two-tailed paired Student’s t test. ns, nonsignificant. (I) Protein expression of Integrin β1 and the indicated EV markers in the UC EV protein extract; average densitometric values of at least three independent biological replicates were normalized to siCtrl. (J) Representative TEM micrographs of EVs enriched by ultracentrifugation (UC EVs) from control or ErbB3-depleted MCF10A cells and stained with gold-conjugated anti-CD63 antibody (CD63-EVs are indicated by red arrows). (K) Cell viability assay measured with PrestoBlue Cell Viability Reagent in MCF10A cells transiently transfected with siCtrl or siErbB3#2 incubated with EV-free media as vehicle (Ctrl) or 1 × 109 nanoparticles of EVs secreted from MCF10A cells transiently transfected with control (VSFsiCtrl) or ErbB3-siRNA (VSFsiErbB3) for 48 h. (L) Matrigel invasion assay in transwells with MCF10A cells stimulated with EV-free media (Ctrl) or incubated with equal number of VSFsiCtrl or VSFsiErbB3 as described in K for 16 h. (M) Matrigel invasion assay in transwells with MCF10A WT cells prestimulated for 48 h with EV-free media (Ctrl) or incubated with equal number of VSFsiCtrl or VSFsiErbB3 in the presence of 5 ug/ml IgG or Integrin β1 monoclonal antibody (mAb; P4C10) for an additional 16 h. The data in K–M are presented as mean values of three biological replicates ±SEM and P values shown based on two-way ANOVA, followed by multiple paired comparisons conducted by means of Bonferroni’s posttest method (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). Scale bars, 15 µm (A), 10 µm (G), 200 nm (J).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal