Figure S4.
ErbB3 does not affect exocytic trafficking of VSV-G but reduces EV release. (A) Immunoblotting analysis of isolated surface-biotinylated VSV-G-ts45-GFP, following its temperature-dependent release from the ER for 2 h in MCF10A cells transiently transfected with control siRNA (siCtrl) or siErbB3#2. Note that ErbB3 depletion does not influence the emergence of VSV-G at the plasma membrane. (B) Confocal imaging of VSV-G-ts45-GFP (green) and markers of the TGN (GM130; blue) and the cell surface (E-cadherin; magenta) in control or ErbB3 siRNA-transfected MCF10A cells at nonpermissive temperature (40°C) and following its subsequent shift to permissive (32°C) temperature. Note that VSV-G-ts45-GFP localizes to the ER at 40°C, while after 30 min at permissive temperature, most of the VSV-G-ts45-GFP has moved to the TGN and after 1 h to the cell surface, both in the presence and in the absence of ErbB3. (C) Representative TEM micrographs of EVs enriched in the VSF of control or ErbB3-depleted MCF10A cells (EVs are indicated by red arrows). (D) Representative nanoparticle traces measured by NTA. The VSF from MCF10A transiently transfected or not (WT) with control siRNA (siCtrl) or independent ErbB3 siRNAs were analyzed by NTA 3.4 software, according to the manufacturer’s protocol. 100-nm beads were used as a positive control. (E) ErbB3 and β-Actin (as a loading control) protein levels in prHMEC cells transiently transfected with the indicated siRNAs. (F and G) EVs enriched in the VSF released by prHMEC transiently transfected with the indicated siRNAs were quantified by NTA in terms of nanoparticle modal size (nm) and number normalized to the total cell number. (H) ErbB3, Rab4, Integrin β1, and β-Actin (as a loading control) protein levels in MCF7 cells transiently transfected with the indicated siRNAs. (I) EVs enriched in the VSF released by MCF7 transiently transfected with the indicated siRNAs were quantified by NTA in terms of modal size (nm) and number of nanoparticles, upon normalization to the total cell number. (J) Representative TEM micrographs of EVs enriched in the VSF of control and ErbB3-depleted or Rab4-depleted MCF7 cells (EVs are indicated by red arrows). Data in F and H are presented as mean values ± SEM. The P value in F was determined by unpaired Student’s t test, while in H, the P value was determined by one-way ANOVA followed by multiple paired comparisons conducted by Bonferroni’s posttest method. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001. (J) Scale bars, 10 µm (B), 200 nm (C), 1 µm (J).

ErbB3 does not affect exocytic trafficking of VSV-G but reduces EV release. (A) Immunoblotting analysis of isolated surface-biotinylated VSV-G-ts45-GFP, following its temperature-dependent release from the ER for 2 h in MCF10A cells transiently transfected with control siRNA (siCtrl) or siErbB3#2. Note that ErbB3 depletion does not influence the emergence of VSV-G at the plasma membrane. (B) Confocal imaging of VSV-G-ts45-GFP (green) and markers of the TGN (GM130; blue) and the cell surface (E-cadherin; magenta) in control or ErbB3 siRNA-transfected MCF10A cells at nonpermissive temperature (40°C) and following its subsequent shift to permissive (32°C) temperature. Note that VSV-G-ts45-GFP localizes to the ER at 40°C, while after 30 min at permissive temperature, most of the VSV-G-ts45-GFP has moved to the TGN and after 1 h to the cell surface, both in the presence and in the absence of ErbB3. (C) Representative TEM micrographs of EVs enriched in the VSF of control or ErbB3-depleted MCF10A cells (EVs are indicated by red arrows). (D) Representative nanoparticle traces measured by NTA. The VSF from MCF10A transiently transfected or not (WT) with control siRNA (siCtrl) or independent ErbB3 siRNAs were analyzed by NTA 3.4 software, according to the manufacturer’s protocol. 100-nm beads were used as a positive control. (E) ErbB3 and β-Actin (as a loading control) protein levels in prHMEC cells transiently transfected with the indicated siRNAs. (F and G) EVs enriched in the VSF released by prHMEC transiently transfected with the indicated siRNAs were quantified by NTA in terms of nanoparticle modal size (nm) and number normalized to the total cell number. (H) ErbB3, Rab4, Integrin β1, and β-Actin (as a loading control) protein levels in MCF7 cells transiently transfected with the indicated siRNAs. (I) EVs enriched in the VSF released by MCF7 transiently transfected with the indicated siRNAs were quantified by NTA in terms of modal size (nm) and number of nanoparticles, upon normalization to the total cell number. (J) Representative TEM micrographs of EVs enriched in the VSF of control and ErbB3-depleted or Rab4-depleted MCF7 cells (EVs are indicated by red arrows). Data in F and H are presented as mean values ± SEM. The P value in F was determined by unpaired Student’s t test, while in H, the P value was determined by one-way ANOVA followed by multiple paired comparisons conducted by Bonferroni’s posttest method. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001. (J) Scale bars, 10 µm (B), 200 nm (C), 1 µm (J).

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