Journal: The Journal of Experimental Medicine

Article Title: A network of immune and microbial modifications underlies viral persistence in the gastrointestinal tract

doi: 10.1084/jem.20191473

Figure Lengend Snippet: The GIT is a long-lived reservoir of LCMV-Cl13 infection. (A and B) Mice were infected with LCMV-Arm (acute) or LCMV-Cl13 (chronic), and 5 and 8 dpi, PFU of virus per gram of tissue or per milliliter of blood plasma was determined. n = 4–5 mice per group from one experiment. *, P < 0.05 by ANOVA test. (B) H&E staining was performed on LI tissue from naive mice or 8 dpi after acute LCMV-Arm or chronic LCMV-Cl13 infection, showing a lack of epithelial or villus pathology during infection. Representative pictures from n = 5 mice per group from one experiment. Scale bars = 50 µm. (C and D) Mice were infected with LCMV-Arm or LCMV-Cl13, and 0, 8, 35, and 100 dpi, the SI, LI, and spleen were analyzed by CyTOF. Graphs show total number of (C) CD45 + viable cells and (D) the indicated immune cell subsets: CD8αβT cells (TCRβ + CD8α + CD8β + ), CD8ααT cells (TCRβ + CD8α + CD8β − ), monocytes/macrophages (CD11b + SiglecF − TCR − B220-CD11c − ), polymorphonuclear cells (CD11b + Ly6G + TCR − B220 − CD11c − ), DCs (CD11c hi MHC-II hi TCR − B220 − ), CD4 T cells (TCRβ + CD4 + ), B cells (B220 + MHC-II + ), ILCs (lin − Thy1.2 + ), and γδT cells (TCRγδ + TCRβ − B220 − ) in the SI, LI, and spleen. *, P < 0.05 (Mann-Whitney test of log-transformed data). n = 10 mice per group. Error bars indicate SEM (A–D).

Article Snippet: The samples were then washed with PBS and pulsed with 12.5 μM cisplatin in PBS for 1 min at RT before quenching with CyTOF staining media (Mg + /Ca 2+ HBSS containing 2% FBS [Multicell], 10 mM Hepes [Corning]) and FBS underlay.

Techniques: Infection, Virus, Clinical Proteomics, Staining, MANN-WHITNEY, Transformation Assay

Journal: Nature cell biology

Article Title: Cathepsin-B-mediated cleavage of Disabled-2 regulates TGF-β-induced autophagy

doi: 10.1038/ncb3388

Figure Lengend Snippet: Dab2 promotes CK2-mediated Beclin-1 phosphorylation resulting in Beclin-1–Vps34 dissociation. (a) Schematic representation of the three CK2 consensus phosphorylation sites in Beclin-1 (Ser64, Ser337 and Ser341). (b) Beclin-1 immunoprecipitation analysis using whole-cell lysates from cells treated with TGF-β for the times indicated. Immunoprecipitated complexes were immunoblotted to detect the expression of Dab2, CK2, Beclin-1, Atg14 and Vps34. Straight immunoblot analysis depicts the expression level of CK2 and Atg14 in whole-cell lysates. (c) Immunoblot analysis of whole-cell lysates from CTSB/OE + LVL Dab2 cells treated with TGF-β for the times indicated. The CK2 inhibitor apigenin (20 µM) was added 6 h before the preparation of whole-cell lysates. (d) In vitro kinase assay using recombinant CK2 and in vitro-synthesized WT Beclin-1 and Beclin-1 mutants S64A, S337A and S341A as substrates. Reactions were subjected to SDS–PAGE and autoradiography. Apigenin was used as a specific inhibitor of CK2. (e) Co-immunoprecipitation analysis using either anti-Flag or anti-Vps34 antibody of whole-cell lysates from NMuMG CTSB/OE cells transfected with Flag-tagged wild-type Beclin-1 and three phosphomimetic Beclin-1 mutants. Immunoprecipitated complexes were immunoblotted with anti-Vps34, anti-Atg14 and anti-Flag antibodies to determine the interaction between Vps34, Atg14 and Beclin-1. (f) Flag immunoprecipitation using whole-cell lysates from 7-day-TGF-β-treated CTSB/OE+LVL Dab2 cells transfected with Flag-tagged wild-type Beclin-1 and its mutants. Immunoprecipitated complexes were immunoblotted to determine the interaction between Dab2, CK2, Vps34, Atg14 and Flag-tagged-Beclin-1. (g) Immunoblot analysis of whole-cell lysates prepared from TGF-β-treated NMuMG LVL Dab2 cells silenced for endogenous Beclin-1 and re-expressing either Flag-tagged wild-type or the indicated Beclin-1 phosphomutants. Blots were probed with the autophagic markers (LC3B and p62) and apoptosis markers (Bim and cleaved caspase-3). Hsp90 was used as a loading control. (h) Immunoblot analysis of whole-cell lysates from NMuMG LVL Dab2 and Beclin-1 KD cells stably re-expressing Flag-tagged exogenous WT or the indicated Beclin-1 mutants. All experiments were repeated at least three times and similar results were observed. Unprocessed original scans of blots are available in Supplementary Fig. 9.

Article Snippet: An autophagic vacuole staining solution Cyto-ID-green detection reagent (Enzo Life Sciences), or antibody against Annexin V (FITC-conjugated, Pharmingen) and PI (BD Pharmingen) were added according to the manufacturers’ instructions and incubated on ice for 30 min. Flow cytometric analysis was performed using a FACS LSRII system (BD Biosciences), and the data were analysed using FlowJo software.

Techniques: Immunoprecipitation, Expressing, Western Blot, In Vitro, Kinase Assay, Recombinant, Synthesized, SDS Page, Autoradiography, Transfection, Stable Transfection

Journal: Nature cell biology

Article Title: Cathepsin-B-mediated cleavage of Disabled-2 regulates TGF-β-induced autophagy

doi: 10.1038/ncb3388

Figure Lengend Snippet: Dab2 promotes chemotherapy-drug-induced cell death by attenuating drug-induced autophagy. (a) Immunoblot analysis of whole-cell lysates prepared from BT-20 and MDA-MB-468 cells, and their modified derivatives, depicting the expression levels of Dab2, CTSB, endogenous Beclin-1 and Flag-tagged muBeclin-1. (b) Clonogenic assays depicting the dose-dependent effects of DOXO on cell growth in NMuMG, BT-20 and MDA-MB-468 cells, and their modified derivatives. (c) Flow cytometric analysis of autophagic flux in NMuMG, BT-20 and MDA-MB-468 cells, and their modified derivatives, incubated in the absence (left peak) or presence (right peak) of DOXO (2.0 µg ml−1) for 24 h. FITC-conjugated Cyto-ID was employed to measure autophagic vacuoles with representative histograms. (d) Flow cytometric analysis of apoptosis using Annexin V staining evaluated in NMuMG, BT-20 and MDA-MB-468 cells, and their modified derivatives, incubated in the absence (top panels) or presence (bottom panels) of DOXO (2.0 µg ml−1). The flow cytometry profile represents Annexin V–FITC staining on the x axis and PI on the y axis. The pink numbers represent the percentages of non-apoptotic cells (lower left quadrant), early apoptotic cells (lower right quadrant), later apoptotic cells (upper right quadrant) and dead cells (upper left quadrant). All experiments were repeated at least three times and similar results were observed. Unprocessed original scans of blots are available in Supplementary Fig. 9.

Article Snippet: An autophagic vacuole staining solution Cyto-ID-green detection reagent (Enzo Life Sciences), or antibody against Annexin V (FITC-conjugated, Pharmingen) and PI (BD Pharmingen) were added according to the manufacturers’ instructions and incubated on ice for 30 min. Flow cytometric analysis was performed using a FACS LSRII system (BD Biosciences), and the data were analysed using FlowJo software.

Techniques: Western Blot, Modification, Expressing, Incubation, Staining, Flow Cytometry