Journal: International Journal of Molecular Sciences

Article Title: The Mutation in wbaP cps Gene Cluster Selected by Phage-Borne Depolymerase Abolishes Capsule Production and Diminishes the Virulence of Klebsiella pneumoniae

doi: 10.3390/ijms222111562

Figure Lengend Snippet: Comparison of virulence of capsule-deficient Kp7De mutant and its parental Kp486 strain. Adhesion ( A ) and time-dependent internalization ( B ) by the A549 lung epithelial cells expressed in CFU per well and shown as the mean ± SEM ( n = 4). ( C ) Phagocytosis by the human monocyte cell line THP1 determined by flow cytometry. UV-killed FITC-labeled bacterial cells were mixed with monocytes at a 100:1 ratio. Quantification of cellular uptake of bacterial cells by phagocytes is expressed as the geometric mean fluorescence intensity (gMFI) from the gated sample with phagocytosing cells ± SEM ( n = 3). ( D ) Time-dependent bactericidal effect of 50% NHS or heat-inactivated NHS for 3 h at 37 °C. The percentage of survival was determined as the number of bacteria that survived relatively to the initial bacterial loading ( n = 3). Asterisks indicate a statistically significant difference ( p < 0.05).

Article Snippet: Briefly, Human monocyte/macrophage cell line THP1 (ATCC, TIB-202) was maintained in RPMI-1640 medium (Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (HIFBS; GIBCO, Life Technologies, Grand Island, NY, USA), 1× glutaMAX (GIBCO, Life Technologies, Grand Island, NY, USA), and 1× antibiotic–antimycotic solution (GIBCO, Life Technologies, Grand Island, NY, USA) at 37 °C in a 5% CO 2 atmosphere. (i) Fluorescence labeling of Klebsiella strains Washed bacteria (ca.

Techniques: Mutagenesis, Flow Cytometry, Labeling, Fluorescence

Journal: Redox Biology

Article Title: Ferroptosis-dependent extracellular vesicles from macrophage contribute to asbestos-induced mesothelial carcinogenesis through loading ferritin

doi: 10.1016/j.redox.2021.102174

Figure Lengend Snippet: CD63-positive ferroptosis-dependent EVs (FedEVs) derived from macrophages simultaneously exposed to asbestos and iron in vivo and in vitro . (a) Histological images of peritoneal wall in situ in the murine model 6 months after ip injection of 3 mg crocidolite; hematoxylin and eosin (HE) image (left upper), Berlin blue staining (insoluble iron as bluish green, left lower), phase-contrast with red fluorescent image (right panels); cell-specific marker: MSLN (mesothelin), mesothelial cell; αSMA (smooth muscle actin), myofibroblast; CD68, macrophage; white arrowheads, crocidolite (bar = 100 μm). (b) High-resolution fluorescent image in granuloma; red, cell specific marker; green, CD63; blue, Hoechst33342 nuclear staining (bar = 20 μm). (c) Quantification of fluorescent intensity of CD63 in different cells surrounding granuloma (n = 30; mean ± SEM). (d) Schematic image of peritoneal wall surrounding the asbestos-induced granuloma. (e, g) Immunoblot analysis of CD63 in cells under ferroptosis and their media; HT1080 fibrosarcoma cells were exposed to RSL3 (10 μM, 24 h), and phorbol myristate acetate (PMA)-primed THP1 macrophage cells were exposed to crocidolite (15 μg/cm 2 ) in the presence of absence of ferric ammonium citrate (Fe, 100 μg/ml) up to 96 h. (f, h) Membrane-catch transmission electronmicroscopy (TEM) images of EVs on carbon grids (bar = 200 nm). (i–l) The precision of nanoparticle tracking analysis (NTA) concentration measurements counted size-distribution of EVs derived from HT1080 (24 h) and THP1 (96 h) in different conditions in i and j. NTA concentration measurements counted number of EVs in k and l (mean ± SEM). Refer to text for details. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MeT-5A human mesothelial cell line (ATCC), THP1 human macrophage cell line and HEK293T cell line (RIKEN Cell Bank) and HT1080 human sarcoma cell line (JCRB cell bank) were grown under standard sterile cell culture conditions (37 °C, humidified atmosphere, 5% CO 2 ) in EMEM containing 10% FBS and 1% antibiotic-antimycotic (Gibco).

Techniques: Derivative Assay, In Vivo, In Vitro, In Situ, Injection, Staining, Marker, Western Blot, Transmission Assay, Concentration Assay

Journal: Redox Biology

Article Title: Ferroptosis-dependent extracellular vesicles from macrophage contribute to asbestos-induced mesothelial carcinogenesis through loading ferritin

doi: 10.1016/j.redox.2021.102174

Figure Lengend Snippet: Mesothelial cells phagocytose FedEVs derived from asbestos-exposed macrophages under ferroptosis. (a) Schema of culture system for the uptake by MeT-5A (recipient) cells of FedEVs from THP1 cells stably expressing GFP-CD63. (b) Fluorescent cellular images of recipient mesothelial cells phagocytosing GFP-positive FedEVs derived from THP1 macrophage cells exposed to asbestos; red, endogenous CD63; bar = 10 μm. (c) Time-course of quantitative colocalization ratio between GFP-CD63 and endogenous CD63 (mean ± SEM). (d, e) FACS analysis of MeT-5A mesothelial cells for catalytic Fe(II) (RhoNox-4, red) after exposure to GFP-CD63-labeled FedEVs derived from HT1080 by ferroptosis-stimulation with RSL3; mean ± SEM). (f, g) FACS analysis of MeT-5A mesothelial cells for catalytic Fe(II) (RhoNox-4, red) after exposure to GFP-CD63-labeled FedEVs derived from THP1 cells by ferroptosis-stimulation with [crocidolite + Fe]; mean ± SEM). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MeT-5A human mesothelial cell line (ATCC), THP1 human macrophage cell line and HEK293T cell line (RIKEN Cell Bank) and HT1080 human sarcoma cell line (JCRB cell bank) were grown under standard sterile cell culture conditions (37 °C, humidified atmosphere, 5% CO 2 ) in EMEM containing 10% FBS and 1% antibiotic-antimycotic (Gibco).

Techniques: Derivative Assay, Stable Transfection, Expressing, Labeling

Journal: Redox Biology

Article Title: Ferroptosis-dependent extracellular vesicles from macrophage contribute to asbestos-induced mesothelial carcinogenesis through loading ferritin

doi: 10.1016/j.redox.2021.102174

Figure Lengend Snippet: Non-selective proteome analysis of asbestos-induced FedEVs reveals ferritins as major components. (a) Proteomaps depicting the fold changes and associated functions for all the identified proteins within FedEVs derived from THP1 macrophage cells (NT or crocidolite + Fe exposure). (b) Relative abundance of proteins in FedEVs (crocidolite + Fe) in comparison to EVs from untreated control THP1 cells. (c) Immunoblot analysis of FedEVs from THP1 macrophage. (d) Quantification of band intensity (n = 4; mean ± SEM). (e) Immunofluorescent analysis of peritoneal wall in situ in the murine model 6 months after ip injection of 3 mg crocidolite; CD63 (FedEVs, green); FtL (ferritin light chain, red) with blue Hoechst33342 nuclear staining (bar = 50 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MeT-5A human mesothelial cell line (ATCC), THP1 human macrophage cell line and HEK293T cell line (RIKEN Cell Bank) and HT1080 human sarcoma cell line (JCRB cell bank) were grown under standard sterile cell culture conditions (37 °C, humidified atmosphere, 5% CO 2 ) in EMEM containing 10% FBS and 1% antibiotic-antimycotic (Gibco).

Techniques: Derivative Assay, Western Blot, In Situ, Injection, Staining

Journal: Redox Biology

Article Title: Ferroptosis-dependent extracellular vesicles from macrophage contribute to asbestos-induced mesothelial carcinogenesis through loading ferritin

doi: 10.1016/j.redox.2021.102174

Figure Lengend Snippet: Mitotic mesothelial cells specifically phagocytosed FedEVs. (a) FedEVs-uptake analysis using MeT-5A mesothelial (recipient) and THP1 macrophage (donor, BFP-CD63 expressed) cells. (b–e) FACS/imaging analysis of mesothelial cells transduced with the Fucci cell cycle sensor; G1 phase, red; S phase, yellow; G2/Mitosis phase, green. Fucci mesothelial cells taken up BFP-CD63-labeled FedEVs after 24 h (mean ± SEM). (e) Live cell imaging of fucci mesothelial cells after 24 h. (f) Immunoblot analysis of Fucci mesothelial cells taken up BFP-CD63-labeled FedEVs after 24 h incubations and sorting; G1, red (Kusabira-orange2) in (b); G2/M: green (Azami-green1) in (b) for cell cycle-associated proteins and iron metabolism-associated proteins. (g) Quantification of band intensity (n = 4; mean ± SEM). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MeT-5A human mesothelial cell line (ATCC), THP1 human macrophage cell line and HEK293T cell line (RIKEN Cell Bank) and HT1080 human sarcoma cell line (JCRB cell bank) were grown under standard sterile cell culture conditions (37 °C, humidified atmosphere, 5% CO 2 ) in EMEM containing 10% FBS and 1% antibiotic-antimycotic (Gibco).

Techniques: Imaging, Transduction, Labeling, Live Cell Imaging, Western Blot

Journal: Redox Biology

Article Title: Ferroptosis-dependent extracellular vesicles from macrophage contribute to asbestos-induced mesothelial carcinogenesis through loading ferritin

doi: 10.1016/j.redox.2021.102174

Figure Lengend Snippet: Phagocytosis of FedEVs causes oxidative DNA base modification (8-hydroxy-2′-deoxyguanosine, 8-OHdG) and DNA double-strand breaks detected by γH2AX in the recipient mesothelial cells. (a) Immunofluorescent images of mesothelial cells after exposure of THP1-derived FedEVs for 24 h; green, CD63-positive FedEVs from THP1 cells; yellow, 8-OHdG; magenda, γH2AX with blue Hoechst33342 nuclear staining (bar = 10 μm). (b–e) FACS analysis, detecting 8-OHdG and γH2AX in mesothelial cells exposed to FedEVs. (f) Immunofluorescent images of mesothelial cells surrounding granuloma in vivo 6 months after ip injection of crocidolite to mice; green, γH2AX; yellow, CD63; magenta, FtL (bar = 20 μm at the left panels; 20 μm at the right 3 panels). (g) Quantification of the γH2AX in mesothelial cells. Refer to text for details (mean ± SEM). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MeT-5A human mesothelial cell line (ATCC), THP1 human macrophage cell line and HEK293T cell line (RIKEN Cell Bank) and HT1080 human sarcoma cell line (JCRB cell bank) were grown under standard sterile cell culture conditions (37 °C, humidified atmosphere, 5% CO 2 ) in EMEM containing 10% FBS and 1% antibiotic-antimycotic (Gibco).

Techniques: Modification, Derivative Assay, Staining, In Vivo, Injection

Journal: Cell Death & Disease

Article Title: Deubiquitination of CD36 by UCHL1 promotes foam cell formation

doi: 10.1038/s41419-020-02888-x

Figure Lengend Snippet: a , b THP1 and RAW264.7 cells were treated with LDN57444 (5, 10, 20 μM) or UCHL1 siRNA (50 nM) for the indicated time. Western blot analysis for SR-A, CD36, SR-B, ABCA1, ABCG1, and Lox-1. c , d Peritoneal macrophages (pMΦ) were treated with UCHL1 siRNA or LDN57444 and cells lysates were subjected to western blot assay for CD36. e The treated cells were subjected to RT-PCR analysis for CD36. f Macrophages were exposed to cycloheximide (CHX) for the indicated times and LDN57444 or UCHL1 siRNA in combination with CHX for the same time. Western blot analysis for CD36 expression. h THP1 cells were exposed to MG132 for 6 h and then added LDN57444 treatment for 24 h. The protein expression of CD36 was tested. GAPDH was as the loading control. g , i The bands of CD36 proteins were calculated. * p < 0.05 versus LDN57444 treatment group.

Article Snippet: Human macrophage cell line THP1, murine macrophage cell line RAW264.7, and HEK293T were purchased from ATCC (Manassaa, VA, USA).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Redox Biology

Article Title: Asbestos conceives Fe(II)-dependent mutagenic stromal milieu through ceaseless macrophage ferroptosis and β-catenin induction in mesothelium

doi: 10.1016/j.redox.2020.101616

Figure Lengend Snippet: Crocidolite induces macrophage necrosis via lysosome-dependent cell death at an early phase and later via ferroptosis. (a, b) Cell viability (WST) and necrosis ratio (LDH) assays reveal higher toxicity of crocidolite than anthophyllite for THP1 macropahge in the presence of excess iron. Crocidolite or anthophyllite asbestos was exposed to THP1 macrophage cells in the presence of 10 μg/ml ferric ammonium citrate (FAC) for 48 h. (c) Transmission electron microscopy of M0-state THP1 macrophage exposed to asbestos for 24 h. Crocidolite causes lysosomal rupture whereas anthopyllite fibers are confined inside lysosome (bar = 5 μm). (d, e) Transmission electron microscopy of mitochondrial damage in THP1 after crocidolite exposure. Significantly shorter mitochondria are observed (bar = 200 nm). NT, no-treatment. (f, g) Nuclear localization of lysosomal cathepsin B (CTSB) in IL-4 stimulated THP1 cells after exposure to crocidolite (25 μg/cm 2 ) and 100 μg/ml FAC. White arrowhead, nucleus; cathepsin B, red; nucleus, cyan (bar = 50 μm). (h) Time-course immunoblot analysis reveals the involvement of ferroptosis. IL-4 stimulated THP1 was exposed to crocidolite (25 μg/cm 2 ). (i) Immunoblot analysis of IL-4 stimulated THP1 72 h after crocidolite exposure (25 μg/cm 2 ) in the presence of various inhibitors. Necrostatin-1; (necroptosis inhibitor, 40 μM), Ferrostain-1; DFO, deferoxamine (ferroptosis inhibitors; 500 nM and 400 μM, respectively), zVAD-FMK (apoptosis inhibitor, 184 μM), z-FA-FMK (lysosome-dependent cell death inhibitor, 20 μM), Pepstatin-A (autophagic cell death inhibitor, 20 μg/ml). (J) LDH assay in IL-4 stimulated THP1 exposed to crocidolite in the same condition as in (I) (means ± SEM; N ≧ 6). Refer to text for details. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Met5A human mesothelial cell line (ATCC), LP9 human mesothelial cell line (Coriell Institute), Met5A-derived stable cell lines expressing Fucci [ ], mouse RAW264 and human THP1 macrophage cell lines and HEK293T cell line (RIKEN Cell Bank, Tsukuba, Japan) were grown under standard sterile cell culture conditions (37 °C, humidified atmosphere, 5% CO 2 ) in RPMI1640 (Wako, Osaka, Japan), containing 10% fetal bovine serum (FBS; Biowest, #S1780; Nuaillé, France) and 1% antibiotic-antimycotic (Gibco).

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Lactate Dehydrogenase Assay