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

Figure 1.

Figure 1

SEVs passively loaded with DNAJB6 suppress polyQ aggregation in vitro

(A) Experimental strategy for assessing the effect of passive loading of DNAJB6 in sEVs on HTT protein levels in HTT-Q74-RFP HEK293T cells. SEVs isolated from NSCs transfected with empty or GFP-DNAJB6 vector are administered to recipient cells expressing HTT-Q74-RFP. After 48 h, the extent of insoluble polyQ aggregation is assessed using western blotting.

(B) Western blot analysis of donor cells and their derived sEVs (20 μg protein per sample per lane). SEVs derived from empty vector-transfected cells show no enrichment of DNAJB6 with respect to donor cells, whereas DNAJB6 in significantly enriched in sEVs derived from GFP-DNAJB6 transfected cells when compared with donor cells. As expected, GFP signal is present only in GFP-DNAJB6 transfected cells and sEVs and is congruent with the DNAJB6 signal. SEV marker CD9 is highly enriched in sEVs compared with donor cells.

(C) Western blot analysis of cells expressing HTT-Q74-RFP treated with control or GFP-DNAJB6 sEVs or left untreated. RFP (agg) indicates insoluble HTT-Q74-RFP aggregates, whereas mRFP (sol) shows the soluble form of HTT-Q74-RFP. Note the presence of GFP-DNAJB6b (50 kDa) in cells (in addition to DNAJB6a and DNAJB6b as designated) incubated with sEVs derived from GFP-DNAJB6b-transfected cells together with suppression of HTT-Q74-RFP aggregation. GAPDH is used as loading control (30 μg protein per sample per lane).

(D) Western blot analysis of HEK293T WT and DNAJB6 KO cells for the presence (WT) and absence (KO) of DNAJB6. (30 μg protein per sample per lane).

(E) Western blot analysis of HEK293T cells expressing HTT-Q74-RFP treated with control or GFP-DNAJB6 sEVs or left untreated. RFP (agg) shows insoluble HTT-Q74-RFP aggregates, whereas RFP (sol) shows soluble HTT-Q74-RFP, blotted with anti-RFP antibody. Note the presence of GFP-DNAJB6 in cells incubated with sEVs derived from GFP-DNAJB6-transfected cells and corresponding suppression of HTT-Q74-RFP aggregation. GAPDH is used as loading control for both the blots (20 μg protein per sample per lane). Cell: whole-cell lysate; sEV: sEV lysate.