Journal: Antioxidants

Article Title: Exo70 Protects Against Memory and Synaptic Impairments Following Mild Traumatic Brain Injury

doi: 10.3390/antiox14060640

Figure Lengend Snippet: Exo70 overexpression reinforces NMDAR synaptic availability upon mTBI induction. Samples from the dorsal hippocampus were analyzed by Western blot. Here, 30 µg of protein samples were resolved in 10% SDS-PAGE and transferred to PVDF membranes. Tubulin and Actin were used as loading controls. ( A ) Characterization of the lentiviral expression in all experimental groups using GFP and HA antibodies. ( B ) Densitometric analysis of HA-Exo70. ( C ) Western blot analyzing NMDAR synaptic localization after mTBI in injected mice. Samples were analyzed using GluN2B p1472, GluN2B p1336, and total GluN2B antibodies. ( D – F ) Densitometric analysis normalized with loading controls before normalization with Sham-GFP control samples. ( G ) Western blot analyzing NMDAR synaptic-signaling-related proteins after mTBI induction in Exo70-overexpressing samples. Samples were analyzed using pCREB and pERK1/2 antibodies. ( H , I ) The graphs show the densitometric analysis normalized with loading controls before normalization with Sham-GFP control samples. White circles indicate individual experiments. Values represent means ± SEM, n = 5 mice per experimental group. Statistical differences were calculated by two-way ANOVA, followed by post hoc Bonferroni’s test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: statistically non-significant difference.

Article Snippet: Third-generation lentiviral particles were packaged by and acquired from Applied Biological Materials (ABM, Canada) based on the lentiviral vector FUG-1D2A-Exo70-W [ ].

Techniques: Over Expression, Western Blot, SDS Page, Expressing, Injection, Control

Journal: bioRxiv

Article Title: The DNA binding protein BCL6 regulates NFκB-controlled endothelial inflammatory gene expression

doi: 10.1101/2022.11.03.514942

Figure Lengend Snippet: TeloHAEC were mock transduced or stably transduced with lentiviral vectors expressing GFP alone, BCL6 open reading frame, GAPDH shRNA, or BCL6 shRNA. Transduced and control cells were treated with TNFα for 4hr, then global mRNA expression was tested by RNA-Seq. (a) Heat map shows relative expression of TNFα induced genes. (b) Graphs show normalized CPM for transcript counts of representative genes. (c) Transduced endothelial cells were tested for cell surface adhesion molecule expression by flow cytometry. Primary human aortic endothelial cells (n=3 biological replicates) were pre-treated with the BCL6 degrader BI-3802, then stimulated with TNFα. Gene expression was measured by RNA-Seq. (d) Heat map shows relative gene expression. (e) Graph shows percent change in transcript abundance comparing BI-3802 treated HAEC to untreated HAEC, after TNFα stimulation. (f) Primary cardiac endothelial cells were tested for cell surface protein expression by flow cytometry. Results are graphed as fold increase in MFI for each marker (n=4-6 biological replicates). Group differences were analyzed by 2-way ANOVA followed by uncorrected Fisher’s LSD.

Article Snippet: For overexpression, endothelial cells were seeded at 40-70% confluence, then infected with third generation lentiviral particles encoding the BCL6 open reading frame (pLenti-BCL6 ORF-mGFP) or GFP alone (Origene).

Techniques: Stable Transfection, Transduction, Expressing, shRNA, RNA Sequencing Assay, Flow Cytometry, Marker

Journal: Acta Neuropathologica

Article Title: Exosomes induce endolysosomal permeabilization as a gateway by which exosomal tau seeds escape into the cytosol

doi: 10.1007/s00401-020-02254-3

Figure Lengend Snippet: Endosomal membrane permeabilization is triggered by exosomes in HEK293T cellular models. a Experimental diagram showing the generation of lentiviral-derived HEK293T cell lines to produce exosomes labeled with mEmerald-tagged CD9 (Eme-CD9), which were used to treat lentiviral HEK293T cells expressing galectin-3 tagged with mCherry (mCherry-Gal3) for 72 h without lipofectamine. b – m Treatment with Eme-CD9 exosomes results in the permeabilization of endosomes in a small fraction of cells. Selected individual cells are outlined with dashed lines. Scale bar is 10 µm for all panels: b – d Low magnification image showing galectin puncta, representing permeabilized endosomes, which are only detected in a limited number of cells (dashed lines, indicated 1, 2, 3), although all cells appear to have internalized Eme-CD9 exosomes. e – g High magnification image showing that cells with galectin puncta contain Eme-CD9 exosomes. h – j Most untreated control cells do not show galectin puncta. k Quantification of the number of galectin puncta per individual cell reveals a strong increase following treatment with Eme-CD9 exosomes. l Individual cells showing exosome-containing puncta that are only detected in treated cells. Error bars represent ± SD for 38 individual cells analyzed from three independent experiments. **** p < 0.0001. m Manders’ colocalization coefficients, with M1 representing the fraction of the exosomal signal (green circles) that colocalizes with mCherry-Gal3. M2 is the fraction of the mCherry-Gal3 signal (red diamonds) colocalizing with the exosomal green fluorophore. n Diagram showing isolation of rTg4510-derived brain exosomes labeled with CellVue claret Far-red dye (CVC-Tg exos) used to treat tau biosensor cells expressing lentiviral mCherry-Gal3. o – q Treatment with CVC-Tg exosomes results in galectin puncta containing internalized exosomes, which showed the ability to induce tau aggregation in the cytosol (bright green signal of tau-YFP). r – t Tau biosensor cell apparently dividing as it harbors two nuclei (Nuc) despite the presence of mCherry-Gal3 puncta and induced tau aggregates in the cytosol. u – w Control cells did not show galectin puncta or tau aggregation. x Galectin puncta per individual cell strongly increase with the treatment with CVC-Tg exosomes. y Exosome-containing puncta are only detected in cells treated with CVC-Tg exosomes. Error bars represent ± SD for 37 individual cells analyzed from three independent experiments. **** p < 0.0001. z Colocalization showing M1 representing the fraction of the exosomal signal (purple dots) that colocalizes with Cherry-Gal3. M2 is the fraction of the mCherry-Gal3 signal (red diamonds) colocalizing with the exosomal far-red fluorophore. aa Comparative quantification of the percentage of cells showing both galectin and exosomal puncta (double positives) in both mCherry-Gal3 lentiviral cell lines described in a and n . Tau biosensor cells (purple column) and HEK293T cells (green column) show a similar percentage of double positives. 76% of tau biosensor cells with double-positive puncta developed tau aggregates, whereas 24% were permeabilized without forming aggregates (white middle column, + Tau- Agg ). bb Quantification of triple positive cells (exosomes + Gal3 + Tau- Agg ) within the double-positive subpopulations (exosomes + Gal3) described in aa . 100% of tau biosensor cells treated with CVC-labeled tau transgenic exosomes were triple positive, supporting that tau aggregation only occurs in the presence of permeabilization. Error bars represent ± SEM for n = 3 of 1,514 cells analyzed. * p < 0.05; **** p < 0.0001; ns not significant

Article Snippet: Lentiviral constructs were used to generate active viral particles by transfecting Lenti-X cells (632180, Takara Bio) with third-generation lentiviral packaging system plasmids [ ].

Techniques: Derivative Assay, Labeling, Expressing, Isolation, Transgenic Assay