Journal: bioRxiv

Article Title: Exosomes exploit the virus entry machinery and pathway to transmit IFN-α-induced antiviral activity

doi: 10.1101/300715

Figure Lengend Snippet: (A) Preincubation with exosomes increased dextran uptake in HepG2 cells. RhoB-dextran (RhoB-DEX) uptake by HepG2 cells pretreated with exosomes (EXO(+)) was analyzed by flow cytometry, and the MFI is normalized to untreated cells (EXO(-)). (B, C) Confocal images (B) or flow cytometry analysis (C) of exosome and dextran internalization by HepG2 cells treated with EIPA. Scale bars: 10 μm. For flow cytometry analysis, MFI (right) are normalized to DMSO-treated cells. (D-F) Confocal images (D) or flow cytometry analysis (E, F) of exosome and dextran internalization by HepG2 cells treated with IPA-3 or rottlerin. Scale bars: 10 μm. For flow cytometry analysis, MFI (right) are normalized to DMSO-treated cells. (G, H) Exosome uptake is independent of Rac1 or Cdc42. Flow cytometry analysis of exosome internalization by HepG2 cells transfected with EGFP-Rac1 DN mutant (G) or EGFP-Cdc42 DN mutant (H), followed by the incubation with PKH26-labeled exosomes. Transfected cells (EGFP+) are gated, and the uptake of exosomes among transfected cells (EGFP+/PKH26+) is analyzed as described above. (I, J) Internalized exosome colocalized with dextran 30 min (I) or 1 h (J) after internalization. The colocalization of Rho-dextran (red) with PKH67-labeled exosomes (green) is analyzed as described above. Scale bars: 5 μm. (K) Blockade of IFN-α-induced anti-HBV activity transmission by EIPA treatment. HepG2.2.15 cells were pretreated with DMSO or EIPA which presented continuously during following incubation with IFN-EXO or Ctrl-EXO. HBV DNA levels in the medium were quantified by qPCR. The error bars indicate the SD. ⋆ P < 0.05, ⋆⋆ P < 0.01, ⋆⋆⋆⋆ P < 0.0001 (Student’s t-test). The data are representative of three independent experiments.

Article Snippet: K44A dynamin-2 pEGFP was a gift from Sandra Schmid (Addgene plasmid # 34687). pcDNA3-EGFP-Cdc42-T17N (Addgene plasmid # 12976) and pcDNA3-EGFP-Rac1-T17N (Addgene plasmid # 12982) were gifts from Gary Bokoch.

Techniques: Flow Cytometry, Transfection, Mutagenesis, Incubation, Labeling, Activity Assay, Transmission Assay

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 (K44A)-GFP to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Western Blot, Phospho-proteomics, Positive Control, Inhibition, Control, Comparison, Live Cell Imaging, Expressing, Staining, Mutagenesis

Journal: Scientific Reports

Article Title: Structural features in the glycine-binding sites of the GluN1 and GluN3A subunits regulate the surface delivery of NMDA receptors

doi: 10.1038/s41598-019-48845-3

Figure Lengend Snippet: The glycine-binding sites in the GluN1 and GluN3A subunits regulate forward trafficking of GluN1/GluN3A receptors ( a,b ) HEK293 cells co-expressing the indicated GluN1-4a and GFP-GluN3A (GluN3A) subunits together with the dynamin-K44A were labelled with primary anti-GFP and secondary antibodies in non-permeabilizing and permeabilizing conditions. The bar graphs show quantification of relative surface expression of the indicated GluN subunit combinations obtained using quantitative colorimetric assay (n = 6); * p < 0.05 relative to GluN1-4a/GluN3A (ANOVA). ( c,e ) Representative confocal microscopy images of the COS-7 cells transfected with the indicated GluN1-4a and GFP-GluN3A (GluN3A) subunits; the anti-GM130 antibody was used to label the GA. (d,f) Summary of the average intensity of GFP-GluN3A (GluN3A) subunit signal colocalised with GM130 over the average intensity of GFP-GluN3A (GluN3A) subunit signal outside GM130 signal, calculated for the indicated GluN subunit combinations (n ≥ 10 cells); * p < 0.05 vs. GluN1-4a/GluN3A; ANOVA).

Article Snippet: K44A HA-dynamin 2 pcDNA3.1 (dynamin-K44A) was a gift from Sandra Schmid (Addgene plasmid # 34685; http://n2t.net/addgene:34685 ; RRID:Addgene_34685).

Techniques: Binding Assay, Expressing, Colorimetric Assay, Confocal Microscopy, Transfection