Journal: Biomaterials

Article Title: Engineered extracellular vesicle-based gene therapy for the treatment of discogenic back pain.

doi: 10.1016/j.biomaterials.2024.122562

Figure Lengend Snippet: Fig. 2. Non-viral transfection of primary mouse fibroblasts with the developmental transcription factor FOXF1 leads to the generation of FOXF1-loaded engineered extracellular vesicles (eEVs). (A) Schematic diagram illustrating how eEVs are derived from donor cells after transfection with a plasmid encoding for the transcription factor FOXF1. qRT-PCR analysis showing (B) robust upregulation of gene expression (mRNA transcripts) and (C) plasmid DNA copy numbers of FOXF1 in donor cells 24 h after transfection compared to pCMV6-transfected cells (control) (n = 3). (D) Nanosight analysis showing an average particle concentration in the order of ~1.3X1011 particles per mL with an average size of ~280 nm for FOXF1 and pCMV6 eEVs (n = 3). (E) qRT-PCR showing that the level of FOXF1 mRNA packed in the eEVs was 4 orders of magnitude higher compared to pCMV6 eEVs (n = 3). (F) Absolute qRT-PCR analysis showing copy numbers of FOXF1 plasmid DNA packed in eEVs compared to pCMV6 eEVs (n = 3). (G) Western blot characterization confirmed the presence of the EV markers CD63 and TSG101, the cytoskeletal marker tubulin in the donor cells, FOXF1 eEVs and pCMV6 eEVs, and the expression of calnexin (endoplasmic reticulum protein) in the donor cells and the pCMV6 eEVs with a reduced expression in FOXF1 eEV formulation. All error bars are shown as standard error of the mean (SEM). *p < 0.05 and **p < 0.001. Two-tail t-test.

Article Snippet: Primary antibodies FOXF1 (Abcam, ab308633) and CD63 (Abcam, ab216130) were used at a concentration of 1:200, TSG101 (Novus Biologicals, NB200-112) at 1:500, Tubulin (Abcam, ab6046) at 1:500, and Calnexin (Proteintech, 10427-2AP) at 1:1000, diluted in TBS-T and 2.5% BSA (FOXF1 antibody was diluted in 5% milk) and incubated overnight at 4 ◦C.

Techniques: Transfection, Derivative Assay, Plasmid Preparation, Quantitative RT-PCR, Gene Expression, Control, Concentration Assay, Western Blot, Marker, Expressing, Formulation

Journal: Journal of Advanced Research

Article Title: Dromedary milk exosomes as mammary transcriptome nano-vehicle: Their isolation, vesicular and phospholipidomic characterizations

doi: 10.1016/j.jare.2015.10.003

Figure Lengend Snippet: Fig. 2 (a) Protein foot printing of dromedary milk exosomes: The exosomes pellets for both mid and late lactation milk samples were loaded in 10% Tris–glycine gel and stained with commassie brilliant blue. Lanes 1 (25 lg) and 2 (10 lg) represent protein foot printing of extracted exosomes from mid lactation milk samples. Lanes 3 and 4, however, represent extracted exosomes from late lactation milk with different loading amounts (20 lg and 10 lg, respectively). It is clear that from lane 2 and lane 4 with equal loading amounts (10 lg each) that the mid lactation exosome has nearly the same protein banding as that at late lactation. Lane M is the Blue Eye Pre stained protein marker (Jena Bioscience). Each lane represents exosomal proteome extracted from single separate milk sample with no pooling. (b) Western blot of exosomal marker TSG101 (Tumor Susceptibility Gene 101 Protein). After SDS– PAGE, TSG101 was detected from other exosomal protein isolated. Lanes 1, 3, and 5 represent exosomes of mid lactation milk samples with loading amount of 10, 30, and 20 lg of protein, respectively. Lanes 2, 4, and 6 represent late lactation milk exosomes with loading amount of 10, 30, and 20 lg of protein, respectively. Lane M: represent protein marker. Unexpected specific bands were obtained at molecular weight 35 KDa instead of 43 KDa. The Western blot represents one run from three runs with similar results.

Article Snippet: To localize the exosome specific marker, Western blotting was performed with TSG101 polyclonal antibody (Novus Biologicals, Littleton Co, USA, Cat. # NBP1-80244) using HRP-conjugate goat anti-rabbit IgG secondary antibody (Novus Biologicals, Littleton Co, mammary transcriptome nano-vehicle: Their isolation, vesicular and phospho10.003 USA; Cat. # NB730-H) and diaminobenzidine as chromogen substrate (Genemed Biotechnologies kit, Inc., San Francisco, CA, USA, Cat. # 10-0006).

Techniques: Staining, Marker, Western Blot, SDS Page, Isolation, Molecular Weight

Journal: Journal of Extracellular Vesicles

Article Title: Function of ceramide transfer protein for biogenesis and sphingolipid composition of extracellular vesicles

doi: 10.1002/jev2.12233

Figure Lengend Snippet: CERT is associated with the MVE and interacts with Tsg101. (a) Confocal photomicrograph of fluorescent labelling of CERT (green) showing partial colocalization/juxtaposition (white) with EEA1 or LBPA (magenta) in N2a cells. Scale bar 5 μm. (b) CERT detected by immunoblot in the same fractions of Alix after EV purification by sucrose gradient ultracentrifugation 0.29–1.75M. (c) Confocal photomicrograph of PLA assay (green) in tdTomato‐CD9 (red) transfected N2a cells indicating complex formation between Tsg101 and CERT (arrow). DAPI (blue) was used to stain the nuclei. Scale bar 5 μm. (d) Binding kinetics of rTsg101 to immobilized rCERT L measured by ELISA. The Michaelis‐Menten constant (Km) equalled 3.59nM with Vmax equal to 22.7nM. (e) Representative photomicrographs of Tsg101 and CERT PLA signals (red), after treatment with vehicle and 10 μM FB1. Scale bar 5 μm. (f) Box and Whiskers plot of 14–23 pictures per condition, of n = 3 independent experiments, showing PLA signals normalized to the number of nuclei (blue). Unpaired t ‐test (* p < 0.05). (g) AlphaFold2 highest‐ranked structure prediction of the complex between (a) the human START domain of CERT (green A) and the human UEV domain of Tsg101 (chain coloured in light blue B). In (b) the coloured polymers based on pLDDT (score = 93.3) and in (c) PAE plot (pTMscore = 0.742). In (d) the 3D structure of the complex (START domain in green A and UEV domain in magenta B) and in (e) the colour surface electrostatic potential of the complex.

Article Snippet: Recombinant full length human Tsg101 (Origene, TP710086) was incubated for 1 h at 37°C in serial dilutions 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μg/ml in incubation buffer composed by 0.1% BSA in washing buffer 100 μl/well.

Techniques: Western Blot, Purification, Transfection, Staining, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Extracellular Vesicles

Article Title: Function of ceramide transfer protein for biogenesis and sphingolipid composition of extracellular vesicles

doi: 10.1002/jev2.12233

Figure Lengend Snippet: List of interactors of CERT L domains associated to EVs biogenesis

Article Snippet: Recombinant full length human Tsg101 (Origene, TP710086) was incubated for 1 h at 37°C in serial dilutions 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μg/ml in incubation buffer composed by 0.1% BSA in washing buffer 100 μl/well.

Techniques: Clone Assay

Journal: Journal of Extracellular Vesicles

Article Title: Function of ceramide transfer protein for biogenesis and sphingolipid composition of extracellular vesicles

doi: 10.1002/jev2.12233

Figure Lengend Snippet: Overexpression of CERT induces EV formation and secretion. (a) Numbers of EVs in the 10K and 100k EV fractions measured by NTA after pcDNA‐hCERT transfection. Box and Whiskers plot represent N = 6/group. (b) Quantification of Tsg101, Alix and Flotillin‐2 by Western blots. Bar graphs represent average ± SEM of N = 6/group. (c) Enrichment of CERT in 100k EV fraction after transfection with pcDNA‐hCERT. Intensities of bands corresponding to CERT were normalized to Flotillin‐2. Bar graph represents average ± SEM with N = 3/group. (d) The 100k EVs numbers measured by NTA after pcDNA‐hCERT transfection and co‐treatment of the N‐SMase2 inhibitor GW4869 (15 μM). Box and Whiskers plot represents N = 4–9/group. One‐way ANOVA, Sidak's posthoc testing (* p < 0.05; *** p < 0.001; **** p < 0.0001). (e) The 100k EV numbers measured by NTA after treatment with D609 (50 μM). Bar graph represents average ± SEM with each N = 3/group. (f) Quantification of CERT by immunoblots after treatment with D609 (50 μM) in EVs and cell lysate. Bar graphs represent average ± SEM with each N = 3/group. (g) Representative photomicrographs of Tsg101 and CERT PLA signals (red), after transfection with control vector (green) or FL‐CERT L ‐GFP (green). Scale bar 5 μm. Box and Whiskers plot of three independent experiments including a total of 16 pictures/condition showing PLA signals normalized to the number of nuclei (blue). Unpaired t ‐test (* p < 0.05).

Article Snippet: Recombinant full length human Tsg101 (Origene, TP710086) was incubated for 1 h at 37°C in serial dilutions 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μg/ml in incubation buffer composed by 0.1% BSA in washing buffer 100 μl/well.

Techniques: Over Expression, Transfection, Western Blot, Control, Plasmid Preparation

Journal: Journal of Extracellular Vesicles

Article Title: Function of ceramide transfer protein for biogenesis and sphingolipid composition of extracellular vesicles

doi: 10.1002/jev2.12233

Figure Lengend Snippet: CERT inhibitor HPA‐12 reduces production and sphingolipid content of EVs. (a) Fluorescent HPA‐12 (HPA‐12‐NBD) chemical structure. Single plane photomicrograph of fluorescence live microscopy of HPA‐12‐NBD‐treated (green) and tdTomato‐CD9 (red) transfected cells showing partial colocalization. Scale bar 10 μm. b) 100k EV numbers measured by NTA and normalized to cells after 24 h treatment with syn ‐HPA‐12 or the less potent stereoisomer anti ‐HPA‐12. Bar graph shows average ± SEM of N = 3/group. One‐way ANOVA, Tukey posthoc testing (* p < 0.05). (c) Quantification of CD81 and Tsg101 by Western blots. Bar graphs show average ± SEM of N = 4/group. One‐way ANOVA, Tukey posthoc testing * p < 0.05. (d) Cer d18:1/16:0 and SM d18:1/16:0 levels measured in the 100k EVs fraction. Bar graphs show average ± SEM with N = 3/group. Unpaired t ‐test (* p < 0.05). (e) 100k EVs numbers measured by NTA and normalized to cells after 24 h treatment with syn ‐HPA‐12 and/or Cer d18:1/16:0. Bar graph shows average ± SEM with N = 3/group. One‐way ANOVA, Tukey's posthoc testing (* p < 0.05). (f) Quantification of CD81, Tsg101 and Alix by Western blots. Bar graphs show average ± SEM of two independent experiments with each N = 4/group. One‐way ANOVA, Tukey's posthoc testing * p < 0.05. (g) Representative photomicrographs of Tsg101 and CERT PLA signals after treatment with vehicle or synHPA‐12 (8 μM). Scale bar 10 μm. (h). Box and Whiskers plot of PLA signals normalized to the number of nuclei of three independent experiments including a total of 12–20 pictures/condition. Unpaired t ‐test (*** p < 0.001).

Article Snippet: Recombinant full length human Tsg101 (Origene, TP710086) was incubated for 1 h at 37°C in serial dilutions 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μg/ml in incubation buffer composed by 0.1% BSA in washing buffer 100 μl/well.

Techniques: Fluorescence, Microscopy, Transfection, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Function of ceramide transfer protein for biogenesis and sphingolipid composition of extracellular vesicles

doi: 10.1002/jev2.12233

Figure Lengend Snippet: Schematic representation of CERT function in EV biogenesis. There are two routes for CERT‐mediated EV biogenesis. The indirect pathway via the trans ‐Golgi network which is regulated by PI4P generated by PI4PIIIβ and involves SM production. This pathway is related to SMS1, SMS2 and N‐Smase‐2 dependent EV formation. The direct pathway is located in ER‐endosomes/MVE contact sites. The enzyme PI4PIIα regulates CERT transfer of ceramide to the MVE. At the MVE, a complex is formed by Tsg101 and CERT, which requires ceramide.

Article Snippet: Recombinant full length human Tsg101 (Origene, TP710086) was incubated for 1 h at 37°C in serial dilutions 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 μg/ml in incubation buffer composed by 0.1% BSA in washing buffer 100 μl/well.

Techniques: Generated