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. 2021 Oct 14;24(11):103282. doi: 10.1016/j.isci.2021.103282

Figure 3.

Figure 3

DNAJB6-enriched sEVs suppress polyQ aggregation in vitro

(A) Schematic representation of experimental strategy for assessing the effect of DNAJB6-enriched sEVs on HTT protein levels in HTT-Q74-GFP HEK293T cells. Purification of sEVs from NSCs expressing XP-GFP or XP-GFP-DNAJB6 followed by their administration to recipient cells expressing HTT-Q74-GFP is depicted. The recipient cells are assessed for aggregate formation with microscopy and filter trap assay 48 h post-sEV addition.

(B) Control cells and cells treated for 48 h with XP-GFP or XP-GFP-DNAJB6 sEVs (20 μg/mL in 200μL of medium) labeled with a lipophilic dye DiI. Note sEVs taken up by recipient cells can be seen as red dots.

(C) Immunoblotting analysis of DNAJB6 in recipient cells left untreated, treated with XP-GFP or XP-GFP-DNAJB6 sEV fractions. GAPDH was used as a loading control. Note the presence of the exogenously delivered DNAJB6 in XP-GFP-DNAJB6 sEV-treated cells (30 μg protein per sample per lane, n = 2).

(D) Immunoblotting analysis of GFP and DNAJB6 in recipient cells left untreated, treated with XP-GFP or XP-GFP-DNAJB6 sEV fractions. Note the presence of GFP and DNAJB6 in XP-GFP-DNAJB6-treated cell lysates at 50kDa, confirming the presence of GFP-DNAJB6 fusion protein (30 μg protein per sample per lane, n = 1).

(E and F) Confocal microscopy images and graphical representation of quantification of polyQ aggregation in HTT-Q74-GFP-expressing cells 48 h after the addition of XP-GFP or XP-GFP-DNAJB6 sEVs (50 μg/mL in 1mL of medium). Untreated cells serve as a control (set at 100%) (n = 3).

(G) Cell viability of cells expressing HTT-Q74-GFP untreated (control), treated with XP-GFP or XP-GFP-DNAJB6 sEVs (n = 3).

(H) Filter trap assay of extracts of cells expressing HTT-Q74-GFP left untreated (control), treated with XP-GFP or XP-GFP-DNAJB6 sEVs (n = 3).

(I) Graphical representation of the quantification of insoluble aggregates based on three independent filter trap assays. Data are represented as mean ± SD of three independent experiments (∗p < 0.05, ∗∗p < 0.01; ns, nonsignificant; ANOVA Tukey's post hoc test). DAPI was used for nuclear staining. XP: XPack; sEV: sEVs. Scale bars: (B) 10 μm and (E) 25 μm.