Supplementary Figure 7: BMDM derived exosomes containing active NOX2 internalize into the neuron via a dynamin dependent mechanism. | Nature Cell Biology

Supplementary Figure 7: BMDM derived exosomes containing active NOX2 internalize into the neuron via a dynamin dependent mechanism.

From: Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons

Supplementary Figure 7: BMDM derived exosomes containing active NOX2 internalize into the neuron via a dynamin dependent mechanism.

A. Bar graph shows the percentage of GFP positive DRG neurons after plating in microfluidic chambers together with Ncf1-GFP electroporated BMDM and after dynasore versus vehicle (DMSO). B-C Z stack fluorescent images showing GFP expression of Ncf13xS/D-GFP after electroporation in LPS-activated BMDM, in macrophages (B), DRG neurites (C). Ncf1-GFP co-localizes with the endosomal marker Rab7 (B) and with Trk (C) (arrows). Scale bar: 25µm (B) and 100µm (C). Data is expressed as mean percentage of GFP+ DRG cells ± s.e.m. vs total DRG neurons on the grooves. N= 3 independent experiments. (**p<0.0088) indicate significant difference versus GFP-DMSO (two-sided Student’s T test). D-E. Dynasore delivered in BMDM conditioned media over DRG neurites after electroporation of GFP or Ncf1-GFP inhibits endocytosis of vesicles in DRG neurites. GFP and Ncf1-GFP co-localize with the endosomal marker Trk. Scale bar: 100µm. F. Bar graph shows the amount of GFP positive vesicles in DRG neurites after Veh or Dynasore (Dyna) pre-incubation. Data is expressed as average number of GFP+ puncta per 200µm of neurite (2-3 neurites per cell; 6-7 cells per well) ± s.e.m. N= 6 wells per condition. (**p<0.01, ***p<0.005) indicate significant difference versus GFP-Veh (ANOVA followed by Bonferroni test, p<0.0001). G. Dynasore delivered in culture media blocks endocytosis into DRG neurites of Alexa 594 labelled transferrin. Scale bar: 100µm. H. (a) BMDM stained with membrane dye PKH67 (green) and nuclear dye Hoechst (blue). (b) BMDM incubated with non-PKH67 stained exosomes. (c-d) BMDM incubated with exosomes stained with PKH67 (d shows the inset from c). (e-f) BMDM incubated with exosomes derived from BMDM that were pre-incubated with PKH67 (f shows the inset from e). Scale bars: 100µm (a, b, c and e) 10µm (d and f). Experiment performed three times with similar results. Source data for A and F are provided in Supplementary Table 5.

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