Fig. 6: Rab13 contributes to the sorting of cargo proteins into AEVs without affecting their secretion.

A Representative confocal immunofluorescence images from normally cultured and serum-starved 293 T cells, which were immunostained for endogenous Rab13 (green) and CD63 (red). Bar = 10 μm. Enlarged insets were presented at the bottom with a bar of 1 μm. B, C The secretion of sEVs and AEVs from wild-type (WT) or Rab13 knockout (KO) 293 T cells, with or without starvation treatment, was determined by NTA. Data are presented as the mean ± SD of three independent experiments. p-values were determined by one-way ANOVA with Dunnett’s multiple comparisons test (B) or unpaired two tailed Student’s t tests (C). ns: no significance. D Western blot for the AEV fraction from serum-starved WT and Rab13-KO 293 T cells. E Representative images of WT and Rab13-KO 293 T cells immunostained for endogenous Rab13 (green), LC3B (red) and p62(red). Bar = 10 μm. Enlarged insets were displayed with the bar = 1 μm. Percentage colocalization of Rab13 with LC3B or /p62 was quantified by determining Pearson’s correlation coefficients providing a measure for the relative degree of co-localization of proteins. For each sample, five cells were quantified using ImageJ software. Three replicate samples from independent experiments were analyzed for each group. p-values were determined by unpaired two tailed Student’s t tests. ***p = <0.0001 (above), ***p = <0.0001 (below). F Confocal images and enlarged insets of LC3B (green) and CD63(red) in serum-starved WT and Rab13-KO 293 T cells. Bar = 10 μm. Scale bars for enlarged insets: 1 μm. G Representative images of TEM from 293 T cells after starving for 24 h. The green arrows represent the compact multivesicular structure and the yellow arrows represent intraluminal vesicles (ILVs). Bar = 500 nm. Similar results were obtained in three independent experiments. H, I Schematic illustration for flow cytometric sorting of CD63 and LC3B labelled amphisomes. The mCherry-GFP-positive amphisomes (I) were coated onto cover slip intended for confocal imaging. Bar = 2 μm. H Created in BioRender. Ruan, H. (2025) https://BioRender.com/98rha9d. J The secreted sEVs were isolated from 293 T cells co-transfected with CD63-mCherry and LC3B-GFP, and then imaged under super-resolution microscopy. Bar = 200 nm. Similar results were obtained in three independent experiments. K The number of amphisomes in 293 T cells under normal or starved culture conditions, with or without BafA1 treatment, was calculated using flow cytometric analysis. Data are presented as the mean ± SD of three independent experiments. p-values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. ***p = <0.0001, *p = 0.024. L A volcano plot of the changed proteins identified in WT and Rab13-KO 293 T cells. The proteins were plotted according to their -log₁₀ P values as determined by two-tailed t test and log₂ fold change. Each group contained three independent biological replicates. M GO enrichment analysis of decreased proteins in AEV fraction from serum-starved Rab13-KO 293 T cells relative to AEV fraction from serum-starved WT 293 T cells with the terms for biological processes. Source data are provided as a Source Data file.