Journal: Toxins

Article Title: Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

doi: 10.3390/toxins3030218

Figure Lengend Snippet: PMTb-GFP initially colocalizes with CTxB-594, but then diverges and is trafficked to and translocated from acidic endosomes. HEK 293-T cells were transiently transfected with dual SRE-luciferase reporter plasmids and pcDNA3-Gα q as described in Methods. Seven h post-transfection cells were treated with ( a ) NH 4 Cl or ( b ) bafilomycin A1 (BAF) at the indicated concentrations for 15 min before treatment with 100 ng/mL rPMT. After 15 h incubation, cells were harvested and assayed for SRE reporter gene activity, as described in the Methods. SRE fold activation was determined by dividing SRE reporter gene activity in PMT-treated cells by SRE reporter gene activity in untreated control cells. Data is expressed as an average of three experiments ± S.D. with each experiment performed in triplicate, where * p < 0.05, ** p < 0.005, and *** p < 0.000005. ( c – j ) Swiss 3T3 cells were co-treated with PMTb-GFP and CT subunit B Alexa Fluor ® -594 conjugate (CtxB-594) for 3 h, as described in Methods, without ( c – f ) or with ( g – j ) pretreatment for 30 min with 30 mM NH 4 Cl. Cells were visualized by confocal microscopy. Panels ( c ) and ( g ), PMTb-GFP. Panels ( d ) and ( h ), CtxB-594. Panels ( e ) and ( i ), merged images of green and red channels. Panels ( f ) and ( j ), corresponding DIC images. Insets: Enlargement of the indicated section of the image, showing co-localization of PMTb-GFP and CtxB-594.

Article Snippet: Stock solutions of 700 nM bafilomycin A1 (BAF) (Alexis Biochemicals), 200 µM cytochalasin D (Sigma), 100 µM nocodazole (Sigma), and 700 µM LY294002 (Alexis Biochemicals) were created by dissolving the inhibitor in DMSO.

Techniques: Transfection, Luciferase, Incubation, Activity Assay, Activation Assay, Confocal Microscopy

Journal: Toxins

Article Title: Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

doi: 10.3390/toxins3030218

Figure Lengend Snippet: A model of PMT entry and trafficking. PMT enters the cell through an Arf6-dependent endocytic pathway that involves both actin and microtubule dynamics as evidenced by inhibition of PMT-mediated SRE activation upon expression of Arf6 mutants (Arf6-CA or Arf6-DN) or treatment with the inhibitors cytochalasinD (CcD) or nocodazole (Noc), respectively. PMT shares a similar route of endocytosis as transferrin (Tfn) and cholera toxin (CT); however, these pathways subsequently diverge. Tfn-bound receptors are trafficked to a Rab5-containing endosome in a PI 3-kinase-dependent vesicle fusion process, which is inhibited by LY294002. CT is trafficked retrograde through the trans-Golgi network (TGN) to the Golgi-ER. PMT is further trafficked through an endocytic vesicle that becomes acidified prior to translocation of PMT to the cytosol. Inhibitors of endosomal acidification, such as NH 4 Cl and bafilomycin A1 (BafA1), block the translocation of PMT, while brefeldin A (BFA) enhances this process by diverting any trafficking of PMT to the TGN instead to the acidified endocytic vesicles.

Article Snippet: Stock solutions of 700 nM bafilomycin A1 (BAF) (Alexis Biochemicals), 200 µM cytochalasin D (Sigma), 100 µM nocodazole (Sigma), and 700 µM LY294002 (Alexis Biochemicals) were created by dissolving the inhibitor in DMSO.

Techniques: Inhibition, Activation Assay, Expressing, Translocation Assay, Blocking Assay

Journal: EMBO Reports

Article Title: Multinucleation resets human macrophages for specialized functions at the expense of their identity

doi: 10.15252/embr.202256310

Figure Lengend Snippet: Venn diagram showing the commonly upregulated transcripts ( n = 66) in LGCs, FBGCs and osteoclasts (in gray). KEGG and BioPlanet 2019 pathway analyses on the 66 commonly upregulated genes. Pathways in red are connected. Macrophage fusion and multinucleation causes an increased lysosomal compartment and cell metabolism. The commonly upregulated genes in LGCs, FBGCs and osteoclasts ( ATP6V1H , ATP6V1D , TFRC and SLC11A2 ) and their role in the lysosome‐iron pathway. The effect of lysosomotropic agents (hydroxychloroquine, hydroxy S; ammonium chloride, NH 4 Cl) and v‐ATPase inhibitors (Bafilomycin A1, Baf A1; Concanamycin A, Con A) on fusion and multinucleation in LGCs (upper panel), FBGCs (middle panel) and osteoclasts (lower panel). To test the effect of lysosome dysfunction on cellular iron, FeCl 3 was supplemented. Error bars are median with interquartile range; significance tested by one‐way Anova followed by Sídák's multiple comparisons on log transformed data; n = 4 donors; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Simultaneously, cells were incubated with either Bafilomycin A1 (Baf A1, Sanbio B.V., Cat # 11038‐500), Concanamycin A (Con A, Sigma‐Aldrich, Cat # 27689), Hydroxychloroquine sulfate (Hydroxy Sulfate, Sigma‐Aldrich, Cat # 379409) and Ammonium chloride (NH4Cl, Sigma‐Aldrich, Cat # A9434).

Techniques: Transformation Assay

Journal: Theranostics

Article Title: Aichi virus 3C protease modulates LC3- and SQSTM1/p62-involved antiviral response

doi: 10.7150/thno.47077

Figure Lengend Snippet: LC3 and P62 contribute to retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) activity. (A) A549 cells were treated with HCQ and BAF-A1 for 24 h before AiV (MOI 5) infection. After 24 h infection, AiV-infected cells were detected by immunofluorescence assay with anti-VP1 antibody (green). DAPI staining (blue) showed the nuclear location. (B) Plaque assay of medium harvested from the AiV-infected A549 cells with HCQ or BAF-A1 pretreatment, the calculated virus titers are shown. (C) Luciferase reporter assay of IFNβ, NFκB, AP-1 and ISRE in A549 cells (1×10 5 ). Cells were treated with HCQ (50 μM) and BAF-A1 (100 nM) for 24 h followed by dual luciferase assay. (D-E) Immunoblotting assay of RLR signaling and LC3, p62 proteins in A549 cells, which were treated with HCQ (100 nM) and BAF-A1 (100 nM). (F) Protein level changes at 24 h from panels d-e. The data were evaluated by comparison with mock or untreated control cells. +, increase; +/-, modest increase; -, no change. (G-H) LC3 and p62 overexpression induced IFNβ and NFκB luciferase reporter activities. (I-J) Cell lysates harvested from A549 cells with LC3 and p62 overexpression were immunoblotted with the antibodies for the indicated proteins. Data are presented as mean±SD from three independent tests; **, P < 0.01, ***, P < 0.005 vs untreated group.

Article Snippet: The reagents used in this study were rapamycin and hydroxychloroquine (HCQ) (Sigma-Aldrich, St. Louis, MO, USA); 3-Methyladenine (3-MA), Torine 1 and bafilomycin A1 (BAF-A1) (Merck Millipore, Burlington, MA, USA).

Techniques: Activity Assay, Infection, Immunofluorescence, Staining, Plaque Assay, Virus, Luciferase, Reporter Assay, Western Blot, Comparison, Control, Over Expression