Journal: bioRxiv

Article Title: Multimodal engineering of extracellular vesicles for efficient intracellular protein delivery

doi: 10.1101/2023.04.30.535834

Figure Lengend Snippet: ( A ) Schematic of constructs used for developing the VEDIC system. ( B ) Schematic of intein cleavage and intraluminal cargo release during EV-biogenesis. ( C ) Schematic of fluorescence reporter construct expressed in the reporter cells generated to measure Cre delivery. ( D ) Percentage of GFP positive reporter cells after adding EVs for 2 days, as evaluated by flow cytometry. Unless indicated, the experiments were performed in 96-well plates. ( E ) Fusogen screen in T47D-TL cells after a two-day incubation period with EVs. ( F ) Percentage of GFP positive reporter cells after exposure to EVs derived from VSV-G co-transfected cells. ( G ) EV dose-dependent recombination in B16F10-TL reporter cells mediated by CD63-Intein-Cre+VSV-G EVs. ( H ) Cre and VSV-G protein was detected in T47D-TL reporter cells by Western blot analysis, 48 hours (h) after addition of engineered EVs loaded with Cre in 24-well plates. Two-way ANOVA multiple comparisons test was used for analysis of (F) and (G); One-way ANOVA multiple comparisons test was used for analysis of (E). Data are shown as mean + SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: In a v-bottom 96-well plate, 25 μl of each sample was incubated with APC-labelled CD63 antibody (Miltenyi Biotec, Germany; 1 nM per well) overnight under dark conditions.

Techniques: Construct, Fluorescence, Generated, Flow Cytometry, Incubation, Derivative Assay, Transfection, Western Blot

Journal: bioRxiv

Article Title: Multimodal engineering of extracellular vesicles for efficient intracellular protein delivery

doi: 10.1101/2023.04.30.535834

Figure Lengend Snippet: ( A and B ) VSV-G+ and CD63+ vesicle numbers as determined by single-vesicle flow cytometry after transfection with VSV-G-mNeonGreen alone or VSV-G-mNeonGreen and CD63 together. ( C ) Schematic of constructs generated for developing the VFIC system. ( D ) EV dose-dependent recombination in B16F10-TL cells mediated by VSV-G-Foldon-Intein-Cre and VSV-G-Intein-Cre EVs as evaluated by flow cytometry. ( E ) Representative images showing GFP positive HeLa-TL cells 24, 48 and 72 h after exposure to VFIC EVs at different doses. Scale bar, 100 µm. ( F-I ) Recombination in hard-to-transfect reporter cells (MSC-TL, THP-1-TL, Raw264.7-TL and K562-TL) mediated by VFIC EVs after 48 h. Two-way ANOVA multiple comparisons test was used for analysis of (D), and (F) to (I). Data are shown as mean + SD, * p < 0.05; ** p < 0.01; *** p < 0.001; ns: non-significant.

Article Snippet: In a v-bottom 96-well plate, 25 μl of each sample was incubated with APC-labelled CD63 antibody (Miltenyi Biotec, Germany; 1 nM per well) overnight under dark conditions.

Techniques: Flow Cytometry, Transfection, Construct, Generated

Journal: bioRxiv

Article Title: Multimodal engineering of extracellular vesicles for efficient intracellular protein delivery

doi: 10.1101/2023.04.30.535834

Figure Lengend Snippet: ( A and B ) Properties of the two VSV-G mutants: VSV-G P127D loses the capacity to mediate fusion between the EV-and endosomal membranes and VSV-G K47Q is unable to bind to LDLR on the cell surface. ( C ) Confocal immunofluorescence demonstrating the subcellular distribution of mNG in the presence or absence of wild type VSV-G engineered EVs in Huh7 cells. Scale bar, 20 µm. ( D) Subcellular distribution of mNG in different groups after adding the indicated engineered EVs determined by confocal immunofluorescence. Scale bar, 20 µm. ( E ) Western blot evaluation of protein levels of Cre and VSV-G in HeLa-TL reporter cells after addition of engineered EVs with wild type, P127D or K47Q VSV-G in 24-well plates. ( F - H ) Percentage of GFP positive HeLa-TL, T47D-TL and B16F10-TL cells after adding wild type, P127D or K47Q VSV-G CD63-Intein-Cre EVs, as evaluated by flow cytometry. Two-way ANOVA multiple comparisons test was used for analysis of (F) to (H). Data are shown as mean + SD, * p < 0.05; ** p < 0.01; *** p < 0.001. ns: non-significant.

Article Snippet: In a v-bottom 96-well plate, 25 μl of each sample was incubated with APC-labelled CD63 antibody (Miltenyi Biotec, Germany; 1 nM per well) overnight under dark conditions.

Techniques: Immunofluorescence, Western Blot, Flow Cytometry

Journal: bioRxiv

Article Title: The Small GTPase Rab7 Regulates Release of Mitochondria in Extracellular Vesicles in Response to Lysosomal Dysfunction

doi: 10.1101/2023.02.11.528148

Figure Lengend Snippet: a. Representative Western blot analysis of whole cell lysate (WCL) and extracellular vesicle fractions for EV marker proteins Alix, Tsg101, CD81 and the mitochondrial proteins Tim23 and Tom20. The EVs were obtained by differential centrifugation of conditioned media from mouse embryonic fibroblasts collected over 48 h after treatment with vehicle or Bafilomycin (Baf A1, 50 nM). EVs from 1×10 7 cells were loaded. b. Quantification of proteins in large EVs (n=4-8 biologically independent experiments). c. Venn diagram showing overlap of proteins detected in large and small EVs. d. Gene ontology (GO) analysis of the unique 418 proteins identified in large EV fractions with the top 15 terms for cellular component and biological processes plotted according to −log10False Discovery Rate (FDR). e. Representative Western blot analysis of EV proteins CD63, CD81 and the mitochondrial protein Tim23 in heart lysates and EV fractions isolated from heart tissue. f. Quantification of proteins in large EVs (n=4-5). g. PhAM floxed mice were crossed with Myh6-Cre transgenic mice to generate a mouse line with cardiac specific expression of Mito-Dendra2. Expression of Mito-Dendra2 in myocytes was confirmed by imaging of frozen heart sections and by Western blotting of heart lysates. Scale bar=20μm. h. Representative Western blot analysis of CD63, CD81, Tim23, and Mito-Dendra2 in heart lysates and EV fractions isolated from heart tissue. i. Quantification of proteins in large EVs (n=5). Data are mean±SEM. *p<0.05,**p<0.01,***p<0.001 ****p<0.0001, ns = not significant.

Article Snippet: Antibodies used in this study were: anti-Rab7 (9367S), anti-Alix (2171S), anti-CD81 (Mouse:10037S, human:56039S), anti-Tom20 (42406S), anti-Rab4 (2167S), anti-Rab5 (3547S), anti-Rab9 (5118S), anti-Rab11 (5589S), anti-LC3A/B (4108S), anti-Rab27 (69295S), anti-Arl8b (56085S) from Cell Signaling Technology; anti-SQSTM1/p62 (ab56416), anti-Tsg101 (ab83) from Abcam; anti-Tim23 (11123-1-AP) from Proteintech, anti-CD63 (PA5-92370) from Invitrogen; anti-Mitodendra2 (TA150090) from OriGene; anti-GAPDH (GTX627408) from GeneTex; and Anti-Ubiquitin (P4D1) (sc-8017) from Santa Cruz.

Techniques: Western Blot, Marker, Centrifugation, Isolation, Transgenic Assay, Expressing, Imaging

Journal: bioRxiv

Article Title: The Small GTPase Rab7 Regulates Release of Mitochondria in Extracellular Vesicles in Response to Lysosomal Dysfunction

doi: 10.1101/2023.02.11.528148

Figure Lengend Snippet: a. Representative Western blot of Alix, Tsg101, CD81, and Tim23 protein levels in total cell lysates and EV preparations from Atg5 -/- MEFs. The EVs were obtained by differential centrifugation of conditioned media from WT and Atg5 -/- MEFs collected over 48 h after treatment with vehicle or Bafilomycin A1 (Baf A1, 50 nM). EVs from 1×10 7 cells were loaded. b. Quantification of proteins in large EVs (n=4). c. Western blot analysis of CD63, CD81, Tim23 and LC3 protein levels in heart lysates and extracellular vesicle fractions isolated from heart tissue of Atg7 f/f and Myh6-Cre Atg7 f/f mice. d . Quantification of proteins in large EVs (n=4-6). e. Representative Western blot and quantification of LC3I, LC3II and p62 protein levels in WT and Rab7 -/- MEFs (n=8). f. Representative images of co-localization between CD81 and GFP-LC3 in WT and Rab7 -/- MEFs +/- Baf A1 (50nM for 24hr). g. Pearson’s correlation coefficient (n=3 independent experiments with a total of 90 cells scored). Scale bar = 20 μm. Data are mean±SEM. *p<0.05p<0.001 ****p<0.0001, ns = not significant.

Article Snippet: Antibodies used in this study were: anti-Rab7 (9367S), anti-Alix (2171S), anti-CD81 (Mouse:10037S, human:56039S), anti-Tom20 (42406S), anti-Rab4 (2167S), anti-Rab5 (3547S), anti-Rab9 (5118S), anti-Rab11 (5589S), anti-LC3A/B (4108S), anti-Rab27 (69295S), anti-Arl8b (56085S) from Cell Signaling Technology; anti-SQSTM1/p62 (ab56416), anti-Tsg101 (ab83) from Abcam; anti-Tim23 (11123-1-AP) from Proteintech, anti-CD63 (PA5-92370) from Invitrogen; anti-Mitodendra2 (TA150090) from OriGene; anti-GAPDH (GTX627408) from GeneTex; and Anti-Ubiquitin (P4D1) (sc-8017) from Santa Cruz.

Techniques: Western Blot, Centrifugation, Isolation

Journal: bioRxiv

Article Title: The Small GTPase Rab7 Regulates Release of Mitochondria in Extracellular Vesicles in Response to Lysosomal Dysfunction

doi: 10.1101/2023.02.11.528148

Figure Lengend Snippet: a. Representative Western blot of CD63, CD81 and Tim23 in whole heart lysates and extracellular vesicle fractions from Rab7 f/f and Rab7 f/f MCM heart tissue. b. Quantification of protein levels in large EV fractions (n=4-5) c. Negative stain electron microscopy of large EVs isolated from Rab7 f/f and Rab7 f/f MCM heart tissue. d. Mitochondrial DNA (mtDNA) content in large EVs preparations (n=4-5). e. Representative Western blot of CD81, Tim23, and Mito-Dendra 2 protein levels in whole heart lysates and extracellular vesicle fractions from Rab7 f/f ; Mito-Dendra2 and Rab7 f/f MCM ; Mito-Dendra2 heart tissue. Large and small (total) EVs were isolated from hearts using immunoaffinity capture method. f. Quantification of protein levels in the EV fractions (n=6-7). g. Immunostaining and quantification of CD68-positive cells in Rab7 f/f and Rab7 f/f MCM hearts (n=7), Scale bar=20μm. h. Cardiac macrophage content in Rab7 f/f and Rab7 f/f MCM hearts analyzed by flow cytometry (n=6). i. qPCR analysis for inflammatory markers in Rab7 f/f and Rab7 f/f MCM hearts (n=9). j. Assessment of large EV uptake by RAW 264.7 macrophages. Representative images of cells after incubation (24h) with CFSE-labelled EVs (green). Blue (dapi) and red (Lysotracker Red). Scale bar=10μm. k. Quantification of CFSE-labeled EV uptake by cells (n=5, a minimum of 100 cells were scored in each independent experiment). Data are mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant.

Article Snippet: Antibodies used in this study were: anti-Rab7 (9367S), anti-Alix (2171S), anti-CD81 (Mouse:10037S, human:56039S), anti-Tom20 (42406S), anti-Rab4 (2167S), anti-Rab5 (3547S), anti-Rab9 (5118S), anti-Rab11 (5589S), anti-LC3A/B (4108S), anti-Rab27 (69295S), anti-Arl8b (56085S) from Cell Signaling Technology; anti-SQSTM1/p62 (ab56416), anti-Tsg101 (ab83) from Abcam; anti-Tim23 (11123-1-AP) from Proteintech, anti-CD63 (PA5-92370) from Invitrogen; anti-Mitodendra2 (TA150090) from OriGene; anti-GAPDH (GTX627408) from GeneTex; and Anti-Ubiquitin (P4D1) (sc-8017) from Santa Cruz.

Techniques: Western Blot, Staining, Electron Microscopy, Isolation, Immunostaining, Flow Cytometry, Incubation, Labeling

Journal: bioRxiv

Article Title: The Small GTPase Rab7 Regulates Release of Mitochondria in Extracellular Vesicles in Response to Lysosomal Dysfunction

doi: 10.1101/2023.02.11.528148

Figure Lengend Snippet: a. Representative Western blot of CD63, CD81, and Tim23 in whole heart lysates and cardiac tissue EVs prepared from young (4 month) and aged (24 month) mice using differential centrifugation. b. Quantification of proteins in large EV fractions (n=4). c. Representative Western blot and protein quantification of total (large and small) EVs in hearts using immunoaffinity capture (n=5). d. Representative Western blot of CD63, CD81 and Tim23 in whole heart lysates and cardiac tissue EVs prepared from WT and Lamp2 -/- mice using differential centrifugation. e. Quantification of proteins in large EV fractions (n=6). f. Western blot of heart lysates and cardiac tissue extracellular vesicles (EVs) prepared from control and Danon Disease patients (male and female). g. Overview of Rab7 activation and EV secretion in cells. Data are mean±SEM. *p<0.05, **p<0.01, ns = not significant.

Article Snippet: Antibodies used in this study were: anti-Rab7 (9367S), anti-Alix (2171S), anti-CD81 (Mouse:10037S, human:56039S), anti-Tom20 (42406S), anti-Rab4 (2167S), anti-Rab5 (3547S), anti-Rab9 (5118S), anti-Rab11 (5589S), anti-LC3A/B (4108S), anti-Rab27 (69295S), anti-Arl8b (56085S) from Cell Signaling Technology; anti-SQSTM1/p62 (ab56416), anti-Tsg101 (ab83) from Abcam; anti-Tim23 (11123-1-AP) from Proteintech, anti-CD63 (PA5-92370) from Invitrogen; anti-Mitodendra2 (TA150090) from OriGene; anti-GAPDH (GTX627408) from GeneTex; and Anti-Ubiquitin (P4D1) (sc-8017) from Santa Cruz.

Techniques: Western Blot, Centrifugation, Activation Assay