Fig 1: TDP-43 content in EVs isolated from LCLs from WT controls and heterozygous and homozygous GRN mutation carriers. (A) A representative Western blot showing TDP-43 phosphorylated at Ser409/Ser410 residues (upper panel) or total TDP-43 (middle panel). TSG101 and Alix are also shown. (B) Densitometric analysis of FL p-TDP-43 normalized to the respective TDP-43. (C) Densitometric analysis of the 25 kDa CTFs p-TDP-43 normalized to the respective TDP-43. The graph represents median with interquartile ranges (25–75%). Data are from six independent experiments in three WT and nine GRN-mutated LCLs. Statistical analysis by Mann–Whitney test, Het = heterozygous; Hom = homozygous.
Fig 2: Validation of endogenous cardiovascular extracellular vesicles (EVs) from adult zebrafish.A, Schematic describing the centrifugation steps taken to isolate EV fractions following cell dissociation of adult zebrafish ventricular tissue. B and C, Cryo-EM micrograph of isolated EVs from a pool of ventricles (n=60). C, A panel of 4 higher magnification views of the boxed regions in B. D, Histogram of the size distribution of EVs visualized by cryo-EM. n refers to number of EVs analyzed. E, Typical flow cytometry scatter plots showing the gates used to sort adult cardiac EVs and the controls used to define these gates. F and G, Gaussian distribution of NTA analysis on unsorted EV fractions before and after detergent treatment (F) and after sorting for calcein+ EVs and mCherry+ calcein+ EVs (G). H, Dot blot analysis of protein extracted from FAVS isolated particles, sorted for calcein+ EVs from both nontransgenic and Tg(myl7:HRAS-mCherry) adult ventricular tissue, confirms expression of the EV components Alix and Syntenin. I, TEM-negative stain micrographs of FAVS isolated particles, sorted for mCherry+ calcein+ EVs from Tg(myl7:HRAS-mCherry) adult ventricular tissue. J, Schematic overlay describing the position of the 3 vessels visible in the integrated time series of live imaging of endothelial cell-EVs in the peripheral circulation of an adult Tg(actb2:HRAS-EGFP); Tg(kdrl:mCherry-CAAX) double transgenic fish. White and yellow arrows indicate 2 EC-EVs moving with the blood flow. Scale bars: B, 200 nm; C and I, 50 nm; J, 10 µm.
Fig 3: Validation of endogenous cardiovascular extracellular vesicles (EVs) from larval zebrafish.A, Schematic describing the centrifugation steps taken to isolate EV fractions following cell dissociation of whole zebrafish larvae. B and C, Cryo-EM micrograph of isolated EVs from a pool of whole 3 dpf larvae (n=1200). C, A panel of 4 higher magnification views of the boxed regions in B. D, Histogram of the size distribution of EVs visualized by cryo-EM. n refers to number of EVs analyzed. E, Western blot analysis of protein extracted from the cells (Tg-CL) and crude EV fraction (Tg-EVs) isolated from Tg(kdrl:mCherry-CAAX); Tg(fli:GFP) 6 dpf larvae (n=600). F, Schematic describing the sucrose gradient approach used to more precisely isolate EVs from the crude EV fraction. G, Dot blot analysis of protein extracted from the cells and sucrose gradient fractions isolated from nontransgenic (Non-Tg [n=600]) and Tg(kdrl:mCherry-CAAX) (Tg [n=600]) 6 dpf larvae. H, Typical flow cytometry scatter plots showing the gates used to sort EVs and the controls used to define these gates: An extraction buffer only control plus calcein AM reveals background noise, EVs extracted from Tg(actb2:HRAS-EGFP) fish without calcein AM indicates GFP+ EVs, EVs extracted from nontransgenic (wildtype) fish and labeled with calcein AM defines a calcein+ gate and analysis of EVs extracted from Tg(actb2:HRAS-EGFP) fish with calcein AM labeling identifies a gate of GFP+ calcein+ EVs. Treatment of transgenic EVs labeled with calcein AM plus detergent destroys the majority of EVs, confirming their lipidaceous structure. Similar gating strategies can be used to analyze mCherry+ EVs from Tg(kdrl:mCherry-CAAX) and Tg(myl7:HRAS-mCherry) fish. I, Plot of the number of calcein+ EVs of total events from untreated and detergent treated samples. J, Dot blot analysis of protein extracted from FAVS isolated particles, sorted for both mCherry- and mCherry+ EVs from Tg(kdrl:mCherry-CAAX) 6 dpf larvae, confirms expression of the EV component Alix and absence of Gapdh expression. Scale bars: B, 200 nm; C, 50 nm.
Fig 4: Characterization of extracellular vesicle (EVs) isolated from bone marrow supernatant of high-fat (HFD) or control (CON) diet-fed mice placed on exercise (EX) or sedentary (SED) conditions. (A) EV presence confirmation by mesenchymal stromal cell (MSC) lysate positive control measures of ALIX, Flotillin-2, and TSG101 proteins, (B) Total extracted particle concentration measured by nanoparticle tracking analysis (n = 10 per group), and (C) EV size distribution of injected EVs. # p < 0.05, high-fat diet difference versus control diet (n = 10 per group).
Fig 5: Isolation and characterization of PMSC lipid rafts and LRNVs. (A) Overall schematic of lipid raft isolation and synthesis of LRNVs. (B) Representative image of OptiPrep gradient ultracentrifugation. Box highlights the collection of lipid raft fragments at the 20–30% fraction. (C) Dot plot analysis of caveolin-1 (cav-1), GRASP55, and HSP60 expression at different fractions (1 at 0%, 10 at 35%) of the gradient. (D) Five micrograms of whole cell lysates and lipid raft isolates from the same cell line was resolved by SDS-PAGE, and proteins were visualized using Imperial Protein gel stain. (E) Representative Western blot of whole cell lysates and lipid raft isolates probed for common EV markers (ALIX, TSG101, CD9, CD63), lipid raft marker cav-1, and mitochondrial marker HSP60 as the negative control.
Supplier Page from MilliporeSigma for Anti-ALIX antibody produced in rabbit