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. Author manuscript; available in PMC: 2024 Jun 1.
Published in final edited form as: Nat Cell Biol. 2023 Nov 16;25(12):1860–1872. doi: 10.1038/s41556-023-01269-8

Fig. 6: The EV lipid bilayer-lipid A interaction underlies EV capture of LPS.

Fig. 6:

a–c, LPS binding by EVs isolated from WT and Tlr4−/−Cd14−/− mice and incubated in vitro with FITC-LPS for 45 min followed by SEC re-purification as assessed by the LAL (n=3) (a) and ImageStream (n=3) (b,c) analysis. d,e, Percentage of FITC-LPS+ve EVs (d) and FITC MFI (e) of EVs isolated from FITC-LPS-injected WT and Tlr4−/−Cd14−/− mice (n=3). f–i, LPS binding by EVs preincubated with isotype, anti-HMGB1 (f,g), or anti-LBP (h,i) antibodies (Ab) and incubated with FITC-LPS for 45 min followed by SEC re-purification as assessed by the LAL (n=4) (f,h) and ImageStream (n=3) (g,i) analysis. j,k, LPS binding by unshaved and surface protein-shaved EVs incubated with FITC-LPS followed by SEC re-purification as assessed by the LAL (n=3) (j) and ImageStream (n=3) (k) analysis. l–n, LPS binding by EV mimic liposomes incubated with PBS or biotin-LPS for 45 min as assessed by the LAL (l; n=4) and ImageStream analysis (Percentage of biotin-LPS+ve EV mimic liposomes (m; n=4) and streptavidin-FITC MFI of EV mimic liposomes (n; n=3)). o,p, Percentage of FITC-LPS+ve EV mimic liposomes (o; n=6) and FITC MFI of EV mimics (p; n=5) isolated from mice 1.5 h post-injection with DiD-labeled EV mimics followed by FITC-LPS. q, Cell death in indicated BMDMs stimulated as indicated for 16 h. r–t, The LAL (r) and ImageStream analysis [streptavidin-FITC+ve EVs (s) and streptavidin-FITC-MFI (t)] of EVs incubated with biotin-LPS or biotin-lipid A for 45 min followed by SEC re-purification (n=4). u–x, Lipid A binding by unshaved and surface protein-shaved EVs (u,v) or EV mimic liposomes (w,x) incubated with PBS or lipid A for 45 min followed by SEC re-purification as assessed by the LAL (u,w) and TLR4 reporter (v,x) assays (n=3). Combined data from two (o,p), three (a–e,g,i,j,k,n,q,u–x) or four (f,h,l,m,r,s,t) independent experiments are shown. Horizontal lines represent the mean (a–p,r–x). Data are presented as mean±s.e.m (q). P values were determined by unpaired two-tailed t-test (a-p, r–x) or two-way ANOVA with Sidak’s post-test (q).