Journal: Scientific Reports

Article Title: A translational protocol optimizes the isolation of plasma-derived extracellular vesicle proteomics

doi: 10.1038/s41598-025-08366-8

Figure Lengend Snippet: Size exclusion chromatography using Sepharose CL-4B shows high EV enrichment. ( a ) A set of three peripheral blood samples was each collected in n =4 biological replicates in Streck Cell-Free DNA BCT. In all cases, platelet-poor plasma (PPP) was first processed by density gradient, followed by SEC. After SEC, the EV-enriched fractions were concentrated by ultrafiltration. EV-enriched fractions were analyzed by nanoparticle tracking analysis (NTA), bicinchoninic acid assay (BCA) and western blot (WB). ( b ) Total particle recovery, ( c ) median particle size and ( d ) total amount of protein in EV-enriched fractions after size exclusion chromatography using different SEC resins, measured by ( b ), ( c ) NTA or ( d ) BCA. In ( b )-( d ) symbols signify independent biological replicates. In ( b ) and ( d ) mean of values normalized to Izon columns, is displayed, in ( c ) mean values are displayed for each SEC column. In ( b ) statistical significance is assessed using one sample t test (Bonferroni-Holm-adjusted p-values) with * p =0.0302 (Izon vs. Sepharose) and p =0.1745 (Izon vs. Sephacryl). In ( d ) statistical significance is assessed using one sample t test with * p =0.0182 (Izon vs. Sepharose) and * p =0.0152 (Izon vs. Sephacryl). ( e ) Representative images of standards run through each column to pre-assess their elution profiles. Dextran Blue (2 g/L), high molecular weight polymer, was used to predict the void volume and locate the EV-enriched peak. Thyroglobulin (2 g/L) and bovine serum albumin (BSA, 4 g/L), were used as representatives of a high and low molecular weight proteins, respectively. The signal corresponding to dextran blue was measured by absorbance at 595 nm. For protein quantification Bradford reagent was used and absorbance was measured at 595 nm. ( f ) Representative immunoblotting of 1mL fractions collected from each size exclusion chromatography to asses elution profiles using plasma as starting material. CD9 was selected as EV marker, and ApoB and ApoA1, as lipoprotein markers. Original blots are presented in Supplementary Fig. S12 online. ( g ) CD9/ApoA1 ratio calculated based on immunobloting signal quantification ( n =2 independent biological replicates). Mean values are displayed.

Article Snippet: The following primary antibodies were used for immunoblotting: CD9 (Cell Signalling Technology, #13403, 1:1,000), GAPDH (Proteintech, #60004-1, 1:1,000), ALIX (abcam, ab275377, 1:1,000), ApoA1 (R&D Systems, MAB36641, 1:2,500), ApoB (R&D Systems, MAB41242, 1:1,000), CD235a (Invitrogen, JC159, 1:500), CD41 (Integrin α−IIb, abcam, ab83961, 1:1,000), Hsc70 (abcam, ab51052, 1:2,500).

Techniques: Size-exclusion Chromatography, Clinical Proteomics, Acid Assay, Western Blot, High Molecular Weight, Polymer, Molecular Weight, Marker

Journal: The Journal of Clinical Investigation

Article Title: Somatic mutations in TBX3 promote hepatic clonal expansion by accelerating VLDL secretion

doi: 10.1172/JCI191855

Figure Lengend Snippet: ( A ) qPCR results for genes involved in de novo lipogenesis from Tbx3- KO or -WT mice fed a WD for 6 months. ( B ) qPCR for free fatty acid uptake genes in mice from A . ( C ) qPCR for β-oxidation genes in mice from A . ( D ) qPCR for PC biosynthesis and VLDL secretion genes in mice from A . ( E ) qPCR for PC biosynthesis and VLDL secretion genes in Tbx3- WT or -KO mice fed a WD for 4 weeks. ( F ) qPCR for β-oxidation genes in mice from E . ( G ) Ratio of M+14 myristoylcarnitine to M+16 palmitoylcarnitine in the liver of Tbx3- KO or -WT mice fed a WD for 2 weeks. ( H ) Lipoprotein fractionation (left) and total plasma triglyceride concentration (right) from Tbx3- KO mice fed a WD for 4 weeks. For lipoprotein fractionation, plasma was pooled from 4 mice per group. ( I ) Experimental setup for in vivo VLDL triglyceride secretion assay. ( J ) Plasma triglyceride levels over time from Tbx3 -KO or -WT mice fed a WD for 2 weeks. Tbx3 KO, n = 8; Tbx3 WT, n = 7. ( K ) Quantification of the triglyceride secretion rate from mice from K . ( L ) Plasma concentration of total apolipoprotein B (ApoB) 3 hours after poloxamer injection in mice from K . ( M ) Experimental setup to induce MASLD with a CDA-HFD. ( N ) Liver weight (left), body weight (middle), and liver/body weight ratio (right) of Tbx3 -KO or -WT mice fed a CDA-HFD for 14 weeks. ( O ) Representative H&E images in mice from N . 61. Significance of the difference in plasma triglycerides at 180 minutes after P407 injection in J was calculated using a 2-way ANOVA with Šidák’s post hoc test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bars: 500 μm, left panels; 100 μm, right panels.

Article Snippet: In Tbx3- KO mice, plasma ApoB concentration was measured using a mouse ApoB ELISA kit (Elabscience, E-EL-M3017).

Techniques: Fractionation, Clinical Proteomics, Concentration Assay, In Vivo, Injection

Journal: PLoS Pathogens

Article Title: Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90

doi: 10.1371/journal.ppat.1004424

Figure Lengend Snippet: (A) Comparative s chematic flow diagram of exosome isolation by Exoquick and ultracentrifugation with additional CD63 immuno-magnetic selection. (B) Control uninfected exosomes were mixed with free HCV virus suspension and after 24 hour co-culture, samples were divided into two portions for (1) total RNA extraction or (2) immuno-magnetic CD63 isolation of the exosomes followed by total RNA extraction. Extracted RNA was analysed for HCV RNA content by quantitative real-time PCR. Results are representative of 4 indipendent experiments. (C &D) Exosomes and free HCV virus were isolated as detailed in our methods. Equal numbers of isolated exosomes and free HCV virus were then lysed in RIPA protein extraction buffer. Extracted total protein from exosomes and cell free virus as indicated was subjected to APOE and APOB ELISA analysis according to the manufacturers' protocol. Results are representative of 3 independent experiments with p<0.05 considered statistically significant usisng ANOVA analysis with GraphPad prism 5.0 software.

Article Snippet: Levels of APOE and APOB proteins in the exosomes were identified by using Apolipoprotein E (APOE) Human ELISA Kit (Abcam cat # ab108813) and Human Apolipoprotein B (APOB) Quantikine ELISA Kit (R&D Systems cat # DAPB00) according to the manufacturers' protocols.

Techniques: Isolation, Selection, Control, Virus, Suspension, Co-Culture Assay, RNA Extraction, Real-time Polymerase Chain Reaction, Protein Extraction, Enzyme-linked Immunosorbent Assay, Software