Figure 3.
Loss of ErbB3 directs Integrin β1 for secretion as EV cargo. (A) Confocal imaging of ErbB3-mCherry and indicated Rab marker expressed in MCF10A cells, with or without prior treatment with the recycling inhibitor PQ. (B) Analysis of ErbB3-mCherry colocalization with Rab4 or Rab11. The relative ErbB3 enrichment at the Rab-positive structures was determined by the formula (a−b)/b, where a is the ErbB3-mCherry intensity of the center in Rab4 structures, and b is the adjacent volume (background) for each structure. Each data point represents the minimum average of 20 structures in one cell. P values were determined using unpaired two-tailed Student’s t test. (C) Average projections of all analyzed (indicated number) GFP-Rab4– or GFP-Rab11–positive structures from the indicated number of cells (three independent experiments). (D) Experimental outline of the VSV-G trafficking experiments in MCF10A cells transiently transfected with control siRNA (siCtrl) or siErbB3#2. (E) Immunoblot analysis of the surface pool of VSV-G-ts45-GFP (pull-down of surface-biotinylated VSV-G-ts45-GFP), after its release from the ER at permissive temperature for the indicated times. (F) Quantification of VSV-G-GFP in biotin pull-downs; normalized levels were determined by immunoblot band intensities (n = 3 independent experiments). (G) ErbB3 and β-Actin (as a loading control) protein expression in MCF10A protein extracts of WT and transiently transfected cells with the indicated siRNA. (H) Scheme of the EV enrichment protocol by (1) UC or (2) tangential flow filtration, followed by CD81 immunoaffinity capture of EVs from conditioned media of breast epithelial cells. (I) EVs enriched in the VSF released by the indicated MCF10A cells were quantified by NTA in terms of nanoparticle modal size (nm) and number after normalization to the total cell number. (J and K) Protein expression levels of the indicated EV marker proteins in EV extracts enriched as VSF (J) or CD81-EVs (K) derived from MCF10A cells transiently transfected with the indicated siRNAs; average densitometric values of at least three independent biological replicates were normalized to siCtrl. (L) CD63 protein expression level in EV extracts enriched by UC derived from MCF10A cells transiently transfected with the indicated siRNAs. Densitometric quantification of CD63 expression is presented as the mean ± SEM from three biological replicates, normalized to the corresponding siCtrl condition. (M) Rab4A and GAPDH (as a loading control) protein levels in MCF10A cells transiently transfected with the indicated siRNAs. (N) EVs enriched in the VSF released by MCF10A transiently transfected with the indicated siRNAs were quantified by NTA in terms of nanoparticle modal size (nm) and number upon normalization to the total cell number. Data were presented as mean values ± SEM. P values were determined by two-tailed paired Student’s t test or one-way ANOVA, followed by multiple paired comparisons conducted by means of Bonferroni’s posttest method. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, nonsignificant. Scale bars, 1 µm.

Loss of ErbB3 directs Integrin β1 for secretion as EV cargo. (A) Confocal imaging of ErbB3-mCherry and indicated Rab marker expressed in MCF10A cells, with or without prior treatment with the recycling inhibitor PQ. (B) Analysis of ErbB3-mCherry colocalization with Rab4 or Rab11. The relative ErbB3 enrichment at the Rab-positive structures was determined by the formula (a−b)/b, where a is the ErbB3-mCherry intensity of the center in Rab4 structures, and b is the adjacent volume (background) for each structure. Each data point represents the minimum average of 20 structures in one cell. P values were determined using unpaired two-tailed Student’s t test. (C) Average projections of all analyzed (indicated number) GFP-Rab4– or GFP-Rab11–positive structures from the indicated number of cells (three independent experiments). (D) Experimental outline of the VSV-G trafficking experiments in MCF10A cells transiently transfected with control siRNA (siCtrl) or siErbB3#2. (E) Immunoblot analysis of the surface pool of VSV-G-ts45-GFP (pull-down of surface-biotinylated VSV-G-ts45-GFP), after its release from the ER at permissive temperature for the indicated times. (F) Quantification of VSV-G-GFP in biotin pull-downs; normalized levels were determined by immunoblot band intensities (n = 3 independent experiments). (G) ErbB3 and β-Actin (as a loading control) protein expression in MCF10A protein extracts of WT and transiently transfected cells with the indicated siRNA. (H) Scheme of the EV enrichment protocol by (1) UC or (2) tangential flow filtration, followed by CD81 immunoaffinity capture of EVs from conditioned media of breast epithelial cells. (I) EVs enriched in the VSF released by the indicated MCF10A cells were quantified by NTA in terms of nanoparticle modal size (nm) and number after normalization to the total cell number. (J and K) Protein expression levels of the indicated EV marker proteins in EV extracts enriched as VSF (J) or CD81-EVs (K) derived from MCF10A cells transiently transfected with the indicated siRNAs; average densitometric values of at least three independent biological replicates were normalized to siCtrl. (L) CD63 protein expression level in EV extracts enriched by UC derived from MCF10A cells transiently transfected with the indicated siRNAs. Densitometric quantification of CD63 expression is presented as the mean ± SEM from three biological replicates, normalized to the corresponding siCtrl condition. (M) Rab4A and GAPDH (as a loading control) protein levels in MCF10A cells transiently transfected with the indicated siRNAs. (N) EVs enriched in the VSF released by MCF10A transiently transfected with the indicated siRNAs were quantified by NTA in terms of nanoparticle modal size (nm) and number upon normalization to the total cell number. Data were presented as mean values ± SEM. P values were determined by two-tailed paired Student’s t test or one-way ANOVA, followed by multiple paired comparisons conducted by means of Bonferroni’s posttest method. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns, nonsignificant. Scale bars, 1 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal