Figure 3.
Plekhm1 KO MSCs release syntenin- and syndecan-1–enriched sEVs. (A) Representative immunoblot showing expression of GM130, CD81, and flotillin-1 in WT or Plekhm1 KO MSCs or sEVs derived from equal numbers of WT or Plekhm1 KO MSCs. Vinculin was used as a loading control for MSCs and as a negative marker for sEVs. The data represent results from 4 independent experiments. (B) Quantification of CD81+ particles/mL (×108) in sEVs derived from WT (red) or Plekhm1 KO (blue) MSCs (WT n = 7; Plekhm1 KO n = 8; t test). The values are normalized to the number of donor MSCs. (C) Volcano plot showing protein expression in sEVs from WT vs Plekhm1 KO MSCs. Samples were normalized for protein concentration before mass spectrometry. The x-axis indicates the difference in protein abundance between Plekhm1 KO and WT MSCs, whereas the y-axis indicates the −log P value (Student t test). Proteins in dark red are expressed significantly more highly and in dark green significantly less in sEV from Plekhm1 KO vs WT MSCs. The data represent the results of 4 biological replicates (t test). (D) Representative immunoblot showing expression of syntenin, syndecan-1, and GM130 in WT vs Plekhm1 KO MSCs or sEVs derived from WT or Plekhm1 KO MSCs. The data (syndecan-1) represent results from 4 independent experiments. (E) Representative immunoblot (left) and its quantification (right) showing expression of syntenin and syndecan-1 in WT or Plekhm1 KO MSCs. The values are normalized to the WT for each individual experiment and are the results of 5 (syntenin) or 4 (syndecan-1) biological replicates (multiple t tests). (F) Representative immunofluorescence images of syndecan-1 staining on WT or Plekhm1 KO MSCs (left). The nuclei are counterstained with DAPI (blue) (left). The scale bar depicts 20 μm. The quantification of syndecan-1 fluorescence intensity in WT (red) or Plekhm1 KO (blue) MSCs, normalized to the average value in WT MSCs is shown on the right. The data represent the results of 4 biological replicates (right) (t test). DAPI, 4′,6-diamidino-2-phenylindole.

Plekhm1 KO MSCs release syntenin- and syndecan-1–enriched sEVs. (A) Representative immunoblot showing expression of GM130, CD81, and flotillin-1 in WT or Plekhm1 KO MSCs or sEVs derived from equal numbers of WT or Plekhm1 KO MSCs. Vinculin was used as a loading control for MSCs and as a negative marker for sEVs. The data represent results from 4 independent experiments. (B) Quantification of CD81+ particles/mL (×108) in sEVs derived from WT (red) or Plekhm1 KO (blue) MSCs (WT n = 7; Plekhm1 KO n = 8; t test). The values are normalized to the number of donor MSCs. (C) Volcano plot showing protein expression in sEVs from WT vs Plekhm1 KO MSCs. Samples were normalized for protein concentration before mass spectrometry. The x-axis indicates the difference in protein abundance between Plekhm1 KO and WT MSCs, whereas the y-axis indicates the −log P value (Student t test). Proteins in dark red are expressed significantly more highly and in dark green significantly less in sEV from Plekhm1 KO vs WT MSCs. The data represent the results of 4 biological replicates (t test). (D) Representative immunoblot showing expression of syntenin, syndecan-1, and GM130 in WT vs Plekhm1 KO MSCs or sEVs derived from WT or Plekhm1 KO MSCs. The data (syndecan-1) represent results from 4 independent experiments. (E) Representative immunoblot (left) and its quantification (right) showing expression of syntenin and syndecan-1 in WT or Plekhm1 KO MSCs. The values are normalized to the WT for each individual experiment and are the results of 5 (syntenin) or 4 (syndecan-1) biological replicates (multiple t tests). (F) Representative immunofluorescence images of syndecan-1 staining on WT or Plekhm1 KO MSCs (left). The nuclei are counterstained with DAPI (blue) (left). The scale bar depicts 20 μm. The quantification of syndecan-1 fluorescence intensity in WT (red) or Plekhm1 KO (blue) MSCs, normalized to the average value in WT MSCs is shown on the right. The data represent the results of 4 biological replicates (right) (t test). DAPI, 4′,6-diamidino-2-phenylindole.

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