Fig 2: KLH45-induced LDs co-localize with mitochondria in axons without affecting mitochondrial or Golgi abundance.a, Confocal micrographs of KLH45-treated hippocampal neurons expressing mRuby-synapsin and stained with BODIPY to label LDs. Scale bar: 10 μm. b, Zoomed-in synaptic boutons from (a). Scale bar: 1 μm. c, Confocal micrographs of a straightened 100 µm long segment of hippocampal axon (from Fig. 2a) immunolabeled with anti-synapsin antibody and stained with BODIPY. Merged image and corresponding line intensity profile show repeated overlapping peaks (black arrowheads) of BODIPY (green, LDs) and synapsin (red, synaptic boutons). d, Confocal micrographs of a straightened 50 µm long segment of hippocampal axon (from Fig. 2e) stained with MDH (cyan, LDs) and immunolabeled with anti-TOMM20 (green, mitochondria) and anti-synapsin (red, synaptic boutons) antibodies. Merged image and corresponding line intensity profile show repeated MDH-positive puncta, adjacent to TOMM20 and synapsin peaks (black arrowheads). e, Confocal micrographs of straightened axonal segments from control and KLH45-treated neurons stained with MitoTracker Green. Scale Bar: 5 μm. f, Quantification of mitochondria per 100 μm axonal segment. Data are presented as mean ± SE. p-value (ns, p = 0.68) was determined using unpaired samples two-tailed t-test with n = 24 for Ctrl and n = 23 for KLH45 treated conditions. g, Confocal micrographs of untreated control and KLH45-treated hippocampal neurons immunostained with NeuN (green, neuron) and GM130 (red, cis-Golgi) antibodies. Scale bar: 20 µm. h, Quantification of cis-Golgi network area as a percentage of total neuronal area. Data are presented as mean ± SE. p-value (ns, p = 0.53) was determined using unpaired samples two-tailed t-test with n = 18 for Ctrl and n = 23 for KLH45 treated conditions. i, Confocal micrographs of untreated control, KLH45-treated, and Rotenone-treated hippocampal neurons stained with CellROX to assess ROS level. Rotenon, a mitochondrial complex-I inhibitor that induces ROS production, increases CellROX intensity. Scale bar: 20 μm. j, Quantification of CellROX fluorescence intensity in the soma. Data are presented as mean ± SE. p-values (ns, p = 0.73, ****p < 0.0001) were determined using one-way ANOVA followed by Tukey’s multiple comparison test with n = 10 for Ctrl and KLH45, and n = 11 for Rotenone treated conditions. Source data

Fig 3: Extracellular vesicles accumulate in the liver of S. mansoni‐infected mice. (A) Representative TEM images of EVs isolated from the liver of one representative naïve and one representative S. mansoni‐infected mouse. (B) NTA of liver‐derived EVs measured using the Nanosight LM14C. Representative histograms (left) showing the average size and concentration of liver‐derived EVs isolated from one representative naïve and one representative S. mansoni‐infected mouse. Numbers indicate the mode size; red line indicates means ± SEMs. Pooled quantification (right) of particles per liver analysed (top) and the corresponding mode size (bottom) are shown below. n = 3 – 7; Mean ± SEM. Mann–Whitney U test, *p ≤ 0.05. (C) Proportion of small EVs (<200 nm) and large EVs (>200 nm) in naïve (n = 3) and infected liver‐derived EVs (n = 7), as quantified via NTA from the 100,000 rcf pellet. (D) Count of small and large particles per liver in the 100,000 rcf pellet in naïve and infected liver‐derived EVs. n = 3–7; Mean ± SEM. Mann–Whitney U test, *p ≤ 0.05. (E) Western blot analysis of GM130 and TSG101 expression in naïve and infected liver‐derived EVs, with live (liveT) and apoptotic thymocytes (aT) used as controls. (F) Surface epitope detection of liver‐derived EVs from naïve (grey) or S. mansoni‐infected mice (red) using MACSPlex (left) and flow cytometry (right). Data from two independent experiments are shown; each data point represents a pool of EVs isolated from 6 – 9 livers. Data are plotted as mean ± SEM. (G) CD45+ cell count in the liver of naïve (grey) vs. S. mansoni‐infected (red) mice. n = 5–8; mean ± SEM. Mann–Whitney U test, *p ≤ 0.05. (H) Counts of Annexin V+ cells within different leukocyte populations isolated from the livers of naïve vs. S. mansoni‐infected mice. n = 5 – 8; Mean ± SEM. Multiple unpaired T tests, *p ≤ 0.05. (I) Western blot analysis of cPARP and Histone H3 expression in naïve and infected liver‐derived EVs, and the corresponding bar graphs reporting the ratio to TSG101 signal intensity as analysed via ImageJ. n = 2/condition (6 ‐ 9 livers pooled); Mean ± SEM. (J) Bar graph reporting the read count of mature miRNA detected in EVs from S. mansoni‐infected livers, classified as either murine miRNAs (mmu) or S. mansoni miRNAs (sma). n = 2/condition (6 ‐ 9 livers pooled); Mean ± SEM.