Skip to main content
. 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. 3: Cytosolic delivery of LPS by host-derived EVs.

Fig. 3:

a,b, LPS levels in the cytosol extracted from Casp11−/− BMDMs stimulated for 15 h with purified LPS, PBS-EVs, or LPS-EVs as assessed by the LAL (a) and HEK-Blue TLR4 reporter cell (b) assays (n=3). c,d, Confocal microscopy of Casp11−/− iBMDMs stimulated for 5–6 h with PBS-EVs or FITC-LPS-EVs and stained with anti-CD45 and anti-FITC antibodies to visualize plasma membrane and LPS, respectively (c) and the quantification of FITC intensity (d). e, TEM of Casp11−/− iBMDMs stimulated for 15 h with PBS-EVs, FITC-LPS, or FITC-LPS-EVs and stained with gold-conjugated anti-FITC antibody. Arrows indicate cytosolic localization of LPS. f, LPS levels in the cytosol of splenic myeloid cells from Casp11−/− mice injected with PBS-EVs or FITC-LPS-EVs 5 h post-injection as assessed by the LAL assay (n=3). g,h, ImageStream flow cytometric analysis of intracellular localization of LPS in peritoneal lavage cells of PBS-EV- or FITC-LPS-EV-injected Casp11−/− mice 5 h post-injection stained with anti-CD45 and anti-FITC antibodies (g) to visualize the plasma membrane and LPS, respectively and the quantification of intracellular FITC intensity (h). i, TEM of peritoneal lavage cells isolated from PBS-EV- or FITC-LPS-EV-injected Casp11−/− mice and stained with gold-conjugated anti-FITC antibody. Arrows indicate cytosolic localization of LPS. j,k, ImageStream flow cytometric analysis of intracellular localization of LPS in splenic endothelial cells isolated from PBS-EV- or FITC-LPS-EV-injected Casp11−/− mice 5 h post-injection and stained with anti-CD31 and anti-FITC antibodies (j) to visualize the plasma membrane and LPS, respectively and the quantification of intracellular FITC intensity (k). l, LPS levels in the cytosol of splenic endothelial cells isolated from PBS-EV- or FITC-LPS-EV-injected Casp11−/− mice 5 h post-injection as assessed by the LAL assay (n=4). Combined data from two (c,d,g,h,i) or three (a,b,e,f,j,k,l) independent experiments are shown. Each circle represents a mouse (f,l), cell (d,h,k), or individual experiments (a,b) and the horizontal lines represent the mean. P values were determined by one-way ANOVA with Dunnett’s post-test (a,b) or unpaired two-tailed t-test (d,f,h,k,l). Scale bar, 5 µm (c) or 200 nm (e,i). BF, brightfield.