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  • 95
    Millipore cytochalasin d
    Gliding Motility and Traversal Capacity of Wt and Δ p52  sporozoites. (A) Representative immunofluorescence staining with anti-PfCSP antibodies of the trails produced by Wt and mutant sporozoites deficient in P52 expression (Δ p52-1 and  Δ p52-2 ) as well as Wt sporozoites, treated with cytochalasin D, an inhibitor of sporozoite motility. Characteristic circles of gliding motility are present in Wt and mutant lines, and absent in Wt sporozoites that have been treated with cytochalasin D. (B) Gliding motility of  P. falciparum  Wt (cytochalsin D treated and untreated) and mutant sporozoites as assessed by the capacity to produce the characteristic circles (see A). (C) Cell traversal ability of  P. falciparum  Wt and mutant sporozoites as determined by FACS counting of Dextran positive hepG2 cells. Dex: hepatocytes cultured in the presence of Dextran but without the addition of sporozoites.
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 7585 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Merck KGaA cytochalasin d
    Gliding Motility and Traversal Capacity of Wt and Δ p52  sporozoites. (A) Representative immunofluorescence staining with anti-PfCSP antibodies of the trails produced by Wt and mutant sporozoites deficient in P52 expression (Δ p52-1 and  Δ p52-2 ) as well as Wt sporozoites, treated with cytochalasin D, an inhibitor of sporozoite motility. Characteristic circles of gliding motility are present in Wt and mutant lines, and absent in Wt sporozoites that have been treated with cytochalasin D. (B) Gliding motility of  P. falciparum  Wt (cytochalsin D treated and untreated) and mutant sporozoites as assessed by the capacity to produce the characteristic circles (see A). (C) Cell traversal ability of  P. falciparum  Wt and mutant sporozoites as determined by FACS counting of Dextran positive hepG2 cells. Dex: hepatocytes cultured in the presence of Dextran but without the addition of sporozoites.
    Cytochalasin D, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 95/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Millipore cytoskeletal inhibitors cytochalasin d
    Disruption of <t>cytoskeletal</t> proteins abrogate nanofibrillar collagen-mediated EC alignment and elongation The addition of <t>cytochalasin</t> D (cyto D) or nocodazole (noco) inhibited F-actin assembly on aligned collagen substrates, based on immunofluorescence staining of F-actin or microtubules (A). Quantification of the angle of orientation (B) and cell shape index (C) in the presence of cytoskeleton disruption agents. Arrow denotes orientation of nanofibrils. Scale bar: 100 μm (A). **P
    Cytoskeletal Inhibitors Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore phagocytosis inhibitor cytochalasin d
    B. pseudomallei is killed by NET formation. (A) Purified PMNs pretreated with medium alone, 100 nM the mitogen PMA, 10 μg/ml of the phagocytosis inhibitor <t>cytochalasin</t> D (Cyt D), or 100 units/ml of DNase I after incubation for 90 min from control
    Phagocytosis Inhibitor Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore microfilament inhibitor cytochalasin d
    Neither apoptosis nor necroptosis is triggered by Candidalysin. a Phosphatidylserine exposure and cell viability of hMDMs infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strain ( ece1 Δ/Δ) (MOI 10) or treated with synthetic Candidalysin for 5 h were quantified by staining with FITC-Annexin V and PI, respectively. The number of single-stained or double-stained macrophages was evaluated by manual counting of at least 200 macrophages. b Caspase 3/7 activity was assessed by measuring luminescence of hMDMs 7 h post infection with C . albicans Wt or ece1 Δ/Δ mutant strain (MOI 10) or co-incubation with Candidalysin. Staurosporine served as a positive control. Shown are relative luminescence values (RLU) after background subtraction. c , d LPS-primed hMDMs were treated with synthetic Candidalysin or Nigericin for 5 h. Selected samples were pre-treated with c the necroptosis inhibitor Necrostatin-1 (Nec-1) or d the actin <t>cytoskeleton</t> inhibitor <t>Cytochalasin</t> D or inhibitor solute control DMSO 1 h prior to administration of synthetic Candidalysin or Nigericin. Macrophage lysis was quantified by measuring LDH release. e LPS-primed hMDMs, mBMDMs or mBMDCs were treated with synthetic Candidalysin or Nigericin for 4–5 h. Selected samples were pre-treated with the potassium channel inhibitor glibenclamide or inhibitor solute control DMSO 1 h prior to administration of synthetic Candidalysin or Nigericin. KCl was added after LPS priming. Macrophage lysis was quantified by measuring LDH release. a Data are shown as mean + SD of two different donors. b – e Values are represented as scatterplot with median of three independent donors or experiments ( n ≥ 3). For statistical analysis, a one-way ANOVA with Dunnett’s multiple comparison test was used. *** p ≤ 0.001, significance compared to Candidalysin treatment
    Microfilament Inhibitor Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore actin polymerization cytochalasin d
    Poly(I:C) and TLR3 colocalize at the surface of plasma membrane. HEK293T cells were transfected with TLR3-mCer alone (A) or cotransfected with UNC93B1 (B). Cells were stimulated with rhodamine labeled poly(I:C) (poly(I:C)-R). (B) TLR3 and poly(I:C)-R colocalization was evaluated from plots (right) of fluorescence intensities within 3 μm line profiles (n = 3; i, ii, iii). Three representative speckles where cross-sections were analyzed are marked with the white arrows on the merged image. (C–E) HEK293 cells were transfected with TLR3 alone or with UNC93B1 encoding plasmid. Cells were cotransfected with IFN-β (left) or NF-κB (right) promoter reporter plasmid and Renilla reporter plasmid. Cells were simultaneously treated with poly(I:C) (10 μg/ml) and inhibitors. Cells were treated with increasing amounts (0.2–5 μM) of <t>cytochalasin</t> D (abbr. CD) (C), Dynasore (abbr. Dy) (20–80 μM) (D) or bafilomycin A (abbr. BA) (0.2–2 μM) (E). 8 h after treatment luciferase activity (RLU) was measured in the cell lysates. The results are represented by mean values with SD from triplicate wells. The representative data from three experiments are shown.
    Actin Polymerization Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore actin depolymerizing agent cytochalasin d
    S. enterica serovar Typhimurium induces the formation of actin-dependent membrane protrusions during basolateral invasion of polarized MDCK cells. (A and B) MDCK cells seeded on 3-μm-pore-size filter supports were infected from the basolateral pole with Salmonella and then fixed and processed for transmission electron microscopy (A) or confocal fluorescence microscopy (B). The red areas in panel B represent bacteria, and the green areas represent filamentous actin. The arrowhead in panel B indicates the region magnified in the inset. Bars: 1 μm (A), 10 μm (B). (C) MDCK cells were treated with 10 μg of <t>cytochalasin</t> D (CD)/ml and then subjected to a gentamicin resistance invasion assay. White bars, untreated cells; black bars, cells treated with CD. Results are expressed as mean invasion percentages ± standard deviations. *, P
    Actin Depolymerizing Agent Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore actin depolymerization agent cytochalasin d
    Role of the RhoA/ROCK pathway in leptin-induced GATA-4 translocation. Representative confocal microscopic images of RASMC to study the effect of Y-27632 (Y; 10 μmol/L) or <t>cytochalasin</t> D (Cyto D; 1 μmol/L) on leptin-induced GATA-4 translocation. DAPI stained the nuclei (left panel; blue) and GATA-4 was detected using CruzFluor 488-conjugated secondary antibody (middle panel; green). The overlay (merged) is shown in the right panel. GATA-4 translocation in response to leptin was abolished by Y and Cyto D.
    Actin Depolymerization Agent Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore actin rearrangement inhibitor cytochalasin d
    Bacterial Omp29 up-regulates F-actin rearrangement in OBA9, which results in induction of Aa Y4 entry into OBA9. (A and B) Aa Y4 harvested at mid-log growth was incubated with confluent OBA9 cells (5×10 5 bacteria/100 µl/well) in the presence or absence of <t>cytochalasin</t> D (F-actin rearrangement inhibitor) by the indicated concentration in the figures. The number of bacteria entered or adherent to OBA9 cells was shown (A, bacterial entry; B; bacterial attachment). *, Significantly lower than control medium alone by Student's t test ( P
    Actin Rearrangement Inhibitor Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore f actin destabilizer cytochalasin d
    Bacterial Omp29 up-regulates F-actin rearrangement in OBA9, which results in induction of Aa Y4 entry into OBA9. (A and B) Aa Y4 harvested at mid-log growth was incubated with confluent OBA9 cells (5×10 5 bacteria/100 µl/well) in the presence or absence of <t>cytochalasin</t> D (F-actin rearrangement inhibitor) by the indicated concentration in the figures. The number of bacteria entered or adherent to OBA9 cells was shown (A, bacterial entry; B; bacterial attachment). *, Significantly lower than control medium alone by Student's t test ( P
    F Actin Destabilizer Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore actin polymerization inhibitor cytochalasin d
    Pharmacologic inhibition of cell tension reduces stiffness-dependent differences in cytoskeletal and adhesive architecture U87-MG cells cultured on fibronectin-coated glass and polyacrylamide substrates were treated with 25 μM blebbistatin, 10 μM Y-27632, or 1 μM <t>cytochalasin</t> D for 12–24 hours before fixation and staining for nuclear DNA (blue), F-actin (green), and vinculin (red). In all cases, the number and size of vinculin-positive focal adhesions was reduced with inhibition of cell tension. Blebbistatin and Y-27632 both induced cell spreading on the softest substrates, whereas cytochalasin D induced collapse of the actin cytoskeleton. Bar is 25 μm.
    Actin Polymerization Inhibitor Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore actin cytoskeleton disrupting agent cytochalasin d
    Effects of actin manipulation on oocyte effective tension. (A) Phalloidin staining of a GVI oocyte (left) and a metaphase II (MII) egg (right). Polarity develops during meiotic maturation, such that the metaphase II egg has an actin-rich cap over the meiotic spindle. Red, actin; blue, DNA. Scale bar, (left) 20 μm; (right) 22 μm. (B) Effects on effective tension (T eff ) of actin manipulation by <t>cytochalasin</t> D treatment of GVI oocytes (*p
    Actin Cytoskeleton Disrupting Agent Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore endocytosis inhibitor cyt d
    Effects of actin manipulation on oocyte effective tension. (A) Phalloidin staining of a GVI oocyte (left) and a metaphase II (MII) egg (right). Polarity develops during meiotic maturation, such that the metaphase II egg has an actin-rich cap over the meiotic spindle. Red, actin; blue, DNA. Scale bar, (left) 20 μm; (right) 22 μm. (B) Effects on effective tension (T eff ) of actin manipulation by <t>cytochalasin</t> D treatment of GVI oocytes (*p
    Endocytosis Inhibitor Cyt D, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore microfilament disruptant cytochalasin d
    Effects of actin manipulation on oocyte effective tension. (A) Phalloidin staining of a GVI oocyte (left) and a metaphase II (MII) egg (right). Polarity develops during meiotic maturation, such that the metaphase II egg has an actin-rich cap over the meiotic spindle. Red, actin; blue, DNA. Scale bar, (left) 20 μm; (right) 22 μm. (B) Effects on effective tension (T eff ) of actin manipulation by <t>cytochalasin</t> D treatment of GVI oocytes (*p
    Microfilament Disruptant Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore stress fiber disruptor cytochalasin d
    Effects of <t>cytochalasin</t> D treatment on neural patterning of ECM treated-hiPSCs. The cells treated with ECMs and ± cyto D at day 16 were analyzed for forebrain and hindbrain markers. (A) Representative flow cytometry histograms of forebrain marker
    Stress Fiber Disruptor Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cytochalasin d cd dependent adhesion assay
    Effects of <t>cytochalasin</t> D treatment on neural patterning of ECM treated-hiPSCs. The cells treated with ECMs and ± cyto D at day 16 were analyzed for forebrain and hindbrain markers. (A) Representative flow cytometry histograms of forebrain marker
    Cytochalasin D Cd Dependent Adhesion Assay, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore actin binding protein cytochalasin d
    Random distribution of labeled DNA-strands among daughter nuclei of LRCs. Analysis of the distribution of labeled DNA-strands among daughter nuclei of label-retaining cells (LRCs) on single cell level, using the actin-binding protein cytochalasin D. <t>Cytochalasin</t> D is an actin-binding protein that inhibits cytokinesis, while karyokinesis remains unaffected. Thereby, binucleate cells are created, which enables analysis of the distribution of DNA-strands among daughter nuclei on single cell level. (A): Scheme of the experimental set-up. Animals were pulsed continuously with EdU during embryonic development (day1–day5) and during post-embryonic development (day6–day11), followed by a chase time of 2 months in EdU-free medium. Subsequently animals were soaked in cytochalasin D for 7 days, EdU was visualized and DNA was stained with DAPI. (B): Visualization of binucleate LRCs in macerated cell suspensions. Fluorescence images of EdU (left), DAPI (middle), and interference contrast images (right) of binucleate LRCs, pulsed during embryonic (B1) and post-embryonic (B2, B3) development. Binucleate EdU + cells display equivalent EdU-signal in both daughter nuclei. Abbreviations: EdU, 5-ethynyl-2′-deoxyuridine. Scale bars: 5 µm.
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    Millipore f actin polymerisation inhibitor cytochalasin d
    Random distribution of labeled DNA-strands among daughter nuclei of LRCs. Analysis of the distribution of labeled DNA-strands among daughter nuclei of label-retaining cells (LRCs) on single cell level, using the actin-binding protein cytochalasin D. <t>Cytochalasin</t> D is an actin-binding protein that inhibits cytokinesis, while karyokinesis remains unaffected. Thereby, binucleate cells are created, which enables analysis of the distribution of DNA-strands among daughter nuclei on single cell level. (A): Scheme of the experimental set-up. Animals were pulsed continuously with EdU during embryonic development (day1–day5) and during post-embryonic development (day6–day11), followed by a chase time of 2 months in EdU-free medium. Subsequently animals were soaked in cytochalasin D for 7 days, EdU was visualized and DNA was stained with DAPI. (B): Visualization of binucleate LRCs in macerated cell suspensions. Fluorescence images of EdU (left), DAPI (middle), and interference contrast images (right) of binucleate LRCs, pulsed during embryonic (B1) and post-embryonic (B2, B3) development. Binucleate EdU + cells display equivalent EdU-signal in both daughter nuclei. Abbreviations: EdU, 5-ethynyl-2′-deoxyuridine. Scale bars: 5 µm.
    F Actin Polymerisation Inhibitor Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore signal transduction cytochalasin d
    Eukaryotic cytoskeleton is important for  K. pneumoniae  invasion. Invasion assays of  K. pneumoniae  Ca0438 using Caco-2 cells were performed in the presence of compounds that interfered with host actin (cytochalasin D) or microtubules (nocodazole). Mock invasion in the absence of inhibitors was defined as 100% relative invasiveness. Data are presented as means ± SEM. *,  P
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    Millipore vaginalis
    Human PMNs kill T . <t>vaginalis</t> . (A) Schematic for T . vaginalis cytotoxicity assay: T . vaginalis and PMNs were labelled with CT or CFSE, respectively, and cocultured for 2 hours. Cells were then analyzed by flow cytometry to assess the survival of T . vaginalis . (B) Representative FACS plots of cytotoxicity assay results using an MOI of 0.25 are shown. Surviving T . vaginalis were defined as CT + CFSE − . (C) Percent cytotoxicity of T . vaginalis was determined by counting the number of surviving T . vaginalis as outlined in A and B, at various MOIs, defined as parasite:host cell ratio. All data are represented as mean ± SD. (D) Composite results of T . vaginalis FCSfiles. CFSE, Carboxyfluorescein succinimidyl ester; CT, Cell Tracker; FACS, fluorescence-activated cell sorting; FCS, fluorescence correlation spectroscopy; MOI, multiplicity of infection; PMN, polymorphonuclear cell.
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    Millipore phagocytosis inhibitor
    Human PMNs kill T . <t>vaginalis</t> . (A) Schematic for T . vaginalis cytotoxicity assay: T . vaginalis and PMNs were labelled with CT or CFSE, respectively, and cocultured for 2 hours. Cells were then analyzed by flow cytometry to assess the survival of T . vaginalis . (B) Representative FACS plots of cytotoxicity assay results using an MOI of 0.25 are shown. Surviving T . vaginalis were defined as CT + CFSE − . (C) Percent cytotoxicity of T . vaginalis was determined by counting the number of surviving T . vaginalis as outlined in A and B, at various MOIs, defined as parasite:host cell ratio. All data are represented as mean ± SD. (D) Composite results of T . vaginalis FCSfiles. CFSE, Carboxyfluorescein succinimidyl ester; CT, Cell Tracker; FACS, fluorescence-activated cell sorting; FCS, fluorescence correlation spectroscopy; MOI, multiplicity of infection; PMN, polymorphonuclear cell.
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    Millipore actin polymerization inhibitor
    Human PMNs kill T . <t>vaginalis</t> . (A) Schematic for T . vaginalis cytotoxicity assay: T . vaginalis and PMNs were labelled with CT or CFSE, respectively, and cocultured for 2 hours. Cells were then analyzed by flow cytometry to assess the survival of T . vaginalis . (B) Representative FACS plots of cytotoxicity assay results using an MOI of 0.25 are shown. Surviving T . vaginalis were defined as CT + CFSE − . (C) Percent cytotoxicity of T . vaginalis was determined by counting the number of surviving T . vaginalis as outlined in A and B, at various MOIs, defined as parasite:host cell ratio. All data are represented as mean ± SD. (D) Composite results of T . vaginalis FCSfiles. CFSE, Carboxyfluorescein succinimidyl ester; CT, Cell Tracker; FACS, fluorescence-activated cell sorting; FCS, fluorescence correlation spectroscopy; MOI, multiplicity of infection; PMN, polymorphonuclear cell.
    Actin Polymerization Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore reagents
    Human PMNs kill T . <t>vaginalis</t> . (A) Schematic for T . vaginalis cytotoxicity assay: T . vaginalis and PMNs were labelled with CT or CFSE, respectively, and cocultured for 2 hours. Cells were then analyzed by flow cytometry to assess the survival of T . vaginalis . (B) Representative FACS plots of cytotoxicity assay results using an MOI of 0.25 are shown. Surviving T . vaginalis were defined as CT + CFSE − . (C) Percent cytotoxicity of T . vaginalis was determined by counting the number of surviving T . vaginalis as outlined in A and B, at various MOIs, defined as parasite:host cell ratio. All data are represented as mean ± SD. (D) Composite results of T . vaginalis FCSfiles. CFSE, Carboxyfluorescein succinimidyl ester; CT, Cell Tracker; FACS, fluorescence-activated cell sorting; FCS, fluorescence correlation spectroscopy; MOI, multiplicity of infection; PMN, polymorphonuclear cell.
    Reagents, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore endocytosis inhibitors
    Human PMNs kill T . <t>vaginalis</t> . (A) Schematic for T . vaginalis cytotoxicity assay: T . vaginalis and PMNs were labelled with CT or CFSE, respectively, and cocultured for 2 hours. Cells were then analyzed by flow cytometry to assess the survival of T . vaginalis . (B) Representative FACS plots of cytotoxicity assay results using an MOI of 0.25 are shown. Surviving T . vaginalis were defined as CT + CFSE − . (C) Percent cytotoxicity of T . vaginalis was determined by counting the number of surviving T . vaginalis as outlined in A and B, at various MOIs, defined as parasite:host cell ratio. All data are represented as mean ± SD. (D) Composite results of T . vaginalis FCSfiles. CFSE, Carboxyfluorescein succinimidyl ester; CT, Cell Tracker; FACS, fluorescence-activated cell sorting; FCS, fluorescence correlation spectroscopy; MOI, multiplicity of infection; PMN, polymorphonuclear cell.
    Endocytosis Inhibitors, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dmso
    Human PMNs kill T . <t>vaginalis</t> . (A) Schematic for T . vaginalis cytotoxicity assay: T . vaginalis and PMNs were labelled with CT or CFSE, respectively, and cocultured for 2 hours. Cells were then analyzed by flow cytometry to assess the survival of T . vaginalis . (B) Representative FACS plots of cytotoxicity assay results using an MOI of 0.25 are shown. Surviving T . vaginalis were defined as CT + CFSE − . (C) Percent cytotoxicity of T . vaginalis was determined by counting the number of surviving T . vaginalis as outlined in A and B, at various MOIs, defined as parasite:host cell ratio. All data are represented as mean ± SD. (D) Composite results of T . vaginalis FCSfiles. CFSE, Carboxyfluorescein succinimidyl ester; CT, Cell Tracker; FACS, fluorescence-activated cell sorting; FCS, fluorescence correlation spectroscopy; MOI, multiplicity of infection; PMN, polymorphonuclear cell.
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    Millipore actin depolymerizing agent
    Human PMNs kill T . <t>vaginalis</t> . (A) Schematic for T . vaginalis cytotoxicity assay: T . vaginalis and PMNs were labelled with CT or CFSE, respectively, and cocultured for 2 hours. Cells were then analyzed by flow cytometry to assess the survival of T . vaginalis . (B) Representative FACS plots of cytotoxicity assay results using an MOI of 0.25 are shown. Surviving T . vaginalis were defined as CT + CFSE − . (C) Percent cytotoxicity of T . vaginalis was determined by counting the number of surviving T . vaginalis as outlined in A and B, at various MOIs, defined as parasite:host cell ratio. All data are represented as mean ± SD. (D) Composite results of T . vaginalis FCSfiles. CFSE, Carboxyfluorescein succinimidyl ester; CT, Cell Tracker; FACS, fluorescence-activated cell sorting; FCS, fluorescence correlation spectroscopy; MOI, multiplicity of infection; PMN, polymorphonuclear cell.
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    Millipore endocytyic pathway inhibitors
    Human PMNs kill T . <t>vaginalis</t> . (A) Schematic for T . vaginalis cytotoxicity assay: T . vaginalis and PMNs were labelled with CT or CFSE, respectively, and cocultured for 2 hours. Cells were then analyzed by flow cytometry to assess the survival of T . vaginalis . (B) Representative FACS plots of cytotoxicity assay results using an MOI of 0.25 are shown. Surviving T . vaginalis were defined as CT + CFSE − . (C) Percent cytotoxicity of T . vaginalis was determined by counting the number of surviving T . vaginalis as outlined in A and B, at various MOIs, defined as parasite:host cell ratio. All data are represented as mean ± SD. (D) Composite results of T . vaginalis FCSfiles. CFSE, Carboxyfluorescein succinimidyl ester; CT, Cell Tracker; FACS, fluorescence-activated cell sorting; FCS, fluorescence correlation spectroscopy; MOI, multiplicity of infection; PMN, polymorphonuclear cell.
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    HSV-1 infection of keratinocytes pretreated with inhibitors against microtubules, actin, clathrin, sodium/proton exchanger or with lysosomotropic agents. HaCaT cells or primary human keratinocytes were pretreated with the indicated concentrations of drugs or corresponding solvent controls (C) for 30 min followed by infection with HSV-1 in the presence of the drugs. All infections were at 20 PFU/cell apart from the <t>nocodazole</t> (noc)-treated cells which were infected both at 20 and 5 PFU/cell. All cells were fixed at 2 h p.i. and stained with mouse anti-ICP0. The number of ICP0-expressing cells was determined in at least three independent experiments. The location of ICP0 expression was recorded as only in the nucleus (nuclear ICP0) or in both nucleus and cytoplasm (cytoplasmic ICP0) representing early and early-late phases during infection, respectively. The percentages of infected cells are shown after treatment with nocodazole (noc) in panels A and B, with NH 4 Cl and monensin (mon) in C and D, and with chlorpromazine (CPZ), EIPA and cytochalasin D (CD) in E and F. Results are mean ± standard deviation values. P-values (pair-t-test) are given above the diagram (B, D).
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    HSV-1 infection of keratinocytes pretreated with inhibitors against microtubules, actin, clathrin, sodium/proton exchanger or with lysosomotropic agents. HaCaT cells or primary human keratinocytes were pretreated with the indicated concentrations of drugs or corresponding solvent controls (C) for 30 min followed by infection with HSV-1 in the presence of the drugs. All infections were at 20 PFU/cell apart from the <t>nocodazole</t> (noc)-treated cells which were infected both at 20 and 5 PFU/cell. All cells were fixed at 2 h p.i. and stained with mouse anti-ICP0. The number of ICP0-expressing cells was determined in at least three independent experiments. The location of ICP0 expression was recorded as only in the nucleus (nuclear ICP0) or in both nucleus and cytoplasm (cytoplasmic ICP0) representing early and early-late phases during infection, respectively. The percentages of infected cells are shown after treatment with nocodazole (noc) in panels A and B, with NH 4 Cl and monensin (mon) in C and D, and with chlorpromazine (CPZ), EIPA and cytochalasin D (CD) in E and F. Results are mean ± standard deviation values. P-values (pair-t-test) are given above the diagram (B, D).
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    Millipore anti cnn3 311 325 antibody
    HSV-1 infection of keratinocytes pretreated with inhibitors against microtubules, actin, clathrin, sodium/proton exchanger or with lysosomotropic agents. HaCaT cells or primary human keratinocytes were pretreated with the indicated concentrations of drugs or corresponding solvent controls (C) for 30 min followed by infection with HSV-1 in the presence of the drugs. All infections were at 20 PFU/cell apart from the <t>nocodazole</t> (noc)-treated cells which were infected both at 20 and 5 PFU/cell. All cells were fixed at 2 h p.i. and stained with mouse anti-ICP0. The number of ICP0-expressing cells was determined in at least three independent experiments. The location of ICP0 expression was recorded as only in the nucleus (nuclear ICP0) or in both nucleus and cytoplasm (cytoplasmic ICP0) representing early and early-late phases during infection, respectively. The percentages of infected cells are shown after treatment with nocodazole (noc) in panels A and B, with NH 4 Cl and monensin (mon) in C and D, and with chlorpromazine (CPZ), EIPA and cytochalasin D (CD) in E and F. Results are mean ± standard deviation values. P-values (pair-t-test) are given above the diagram (B, D).
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    N/A
    Cytidine deaminase CDA is an enzyme that scavenges exogenous and endogenous cytidine and 2 deoxycytidine for UMP synthesis This protein is one of several deaminases responsible for maintaining the cellular
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    Image Search Results


    Gliding Motility and Traversal Capacity of Wt and Δ p52  sporozoites. (A) Representative immunofluorescence staining with anti-PfCSP antibodies of the trails produced by Wt and mutant sporozoites deficient in P52 expression (Δ p52-1 and  Δ p52-2 ) as well as Wt sporozoites, treated with cytochalasin D, an inhibitor of sporozoite motility. Characteristic circles of gliding motility are present in Wt and mutant lines, and absent in Wt sporozoites that have been treated with cytochalasin D. (B) Gliding motility of  P. falciparum  Wt (cytochalsin D treated and untreated) and mutant sporozoites as assessed by the capacity to produce the characteristic circles (see A). (C) Cell traversal ability of  P. falciparum  Wt and mutant sporozoites as determined by FACS counting of Dextran positive hepG2 cells. Dex: hepatocytes cultured in the presence of Dextran but without the addition of sporozoites.

    Journal: PLoS ONE

    Article Title: Gene Disruption of Plasmodium falciparum p52 Results in Attenuation of Malaria Liver Stage Development in Cultured Primary Human Hepatocytes

    doi: 10.1371/journal.pone.0003549

    Figure Lengend Snippet: Gliding Motility and Traversal Capacity of Wt and Δ p52 sporozoites. (A) Representative immunofluorescence staining with anti-PfCSP antibodies of the trails produced by Wt and mutant sporozoites deficient in P52 expression (Δ p52-1 and Δ p52-2 ) as well as Wt sporozoites, treated with cytochalasin D, an inhibitor of sporozoite motility. Characteristic circles of gliding motility are present in Wt and mutant lines, and absent in Wt sporozoites that have been treated with cytochalasin D. (B) Gliding motility of P. falciparum Wt (cytochalsin D treated and untreated) and mutant sporozoites as assessed by the capacity to produce the characteristic circles (see A). (C) Cell traversal ability of P. falciparum Wt and mutant sporozoites as determined by FACS counting of Dextran positive hepG2 cells. Dex: hepatocytes cultured in the presence of Dextran but without the addition of sporozoites.

    Article Snippet: Briefly, cytochalasin D (Sigma) was diluted from a 500 µM stock in Me2 SO to a 10 µm final concentration with sporozoites.

    Techniques: Immunofluorescence, Staining, Produced, Mutagenesis, Expressing, FACS, Cell Culture

    Functional validation of the differential coordination between Arp2/3 and VASP in strong accelerating protrusion. a , b t-SNE plots of the denoised protrusion velocity time series of the whole sample overlaid with the density of data. c , d t-SNE plots of the denoised velocities of the sub-clusters (Cluster III-1 and III-2) in Cluster III. e , f Comparison of the proportion of Cluster III-1 and III-2 upon Cytochalasin D treatment ( e ) or CK666 treatment ( f ). The error bars indicate 95% confidence interval of the mean of the cluster proportions. * p

    Journal: Nature Communications

    Article Title: Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging

    doi: 10.1038/s41467-018-04030-0

    Figure Lengend Snippet: Functional validation of the differential coordination between Arp2/3 and VASP in strong accelerating protrusion. a , b t-SNE plots of the denoised protrusion velocity time series of the whole sample overlaid with the density of data. c , d t-SNE plots of the denoised velocities of the sub-clusters (Cluster III-1 and III-2) in Cluster III. e , f Comparison of the proportion of Cluster III-1 and III-2 upon Cytochalasin D treatment ( e ) or CK666 treatment ( f ). The error bars indicate 95% confidence interval of the mean of the cluster proportions. * p

    Article Snippet: For Cytochalasin D experiments, cells were incubated with DMSO or Cytochalasin D (Sigma) for half an hour before imaging.

    Techniques: Functional Assay

    Axonal fragmentation is dependent on actin and myosin dynamics. ( a ) Teased fibers from WT mice sciatic nerves immunostained for neurofilament (Nf-H, green) and stained for actin filaments using phalloidin (red). Both Cytochalasin D (CytD, 2 μ g/ml) and Blebbistatin (Blebb, 500 μ M) treatments inhibits axonal fragmentation at 2 dpi and axonal disintegration at 4 dpi Scale bar, 20 μ m. ( b ) Quantification of the percentage of unfragmented fibers (intact fibers) after each treatment from teased fibers as shown in ( a ). Only at 2 dpi, both CytD and Blebb treatment significantly inhibit fiber fragmentation ( n =3 mice per group; between 80 and 100 fibers per group; * P

    Journal: Cell Death & Disease

    Article Title: Molecular analysis of axonal-intrinsic and glial-associated co-regulation of axon degeneration

    doi: 10.1038/cddis.2017.489

    Figure Lengend Snippet: Axonal fragmentation is dependent on actin and myosin dynamics. ( a ) Teased fibers from WT mice sciatic nerves immunostained for neurofilament (Nf-H, green) and stained for actin filaments using phalloidin (red). Both Cytochalasin D (CytD, 2 μ g/ml) and Blebbistatin (Blebb, 500 μ M) treatments inhibits axonal fragmentation at 2 dpi and axonal disintegration at 4 dpi Scale bar, 20 μ m. ( b ) Quantification of the percentage of unfragmented fibers (intact fibers) after each treatment from teased fibers as shown in ( a ). Only at 2 dpi, both CytD and Blebb treatment significantly inhibit fiber fragmentation ( n =3 mice per group; between 80 and 100 fibers per group; * P

    Article Snippet: The following drugs and concentrations were used: Actinomysin D (Calbiochem, San Diego, CA, USA, #114666, 0,2 μ g/μ l), PD 0325901 (Cayman, Ann Arbor, MI, USA, #13034, 2 μ M), Cytochalasin D (Sigma, #C6637, 2 μ g/ml), Blebbistatin (Sigma, #B0560, 500 μ M), Ciclosporin A (LC Laboratories, Woburn, MA, USA, #C6000, 5 μ M) and Y-27632 (Sigma, #Y0503, 50 μ M).

    Techniques: Mouse Assay, Staining

    Effects of disruption of the actin cytoskeleton with mechanical stimulation through syndecan-4. Shown are Western blot and densitometry analysis of ERK phosphorylation with latrunculin-B and cytochalasin-D treatment under 30 min of strain. The level of ERK phosphorylation in cells that were not chemically treated but mechanically stimulated was statistically higher than both the latrunculin-treated cells ( P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Defining the role of syndecan-4 in mechanotransduction using surface-modification approaches

    doi: 10.1073/pnas.0902639106

    Figure Lengend Snippet: Effects of disruption of the actin cytoskeleton with mechanical stimulation through syndecan-4. Shown are Western blot and densitometry analysis of ERK phosphorylation with latrunculin-B and cytochalasin-D treatment under 30 min of strain. The level of ERK phosphorylation in cells that were not chemically treated but mechanically stimulated was statistically higher than both the latrunculin-treated cells ( P

    Article Snippet: Latrunculin-B (0.4 μg/mL) or cytochalasin-D (0.1 μg/mL; Sigma L5288, Sigma 30385) was added to the cells before the mechanical stimulation.

    Techniques: Western Blot

    WASH-mediated nuclear actin nucleation is needed to assist NURF complex to c-Myc promoter. (A and B) Nuclear and cytoplasmic extracts prepared from sorted LT-HSCs were incubated with recombinant WASH (rWASH; A) or nuclear extracts were incubated with various WASH mutants (B), followed by pyrene actin assays. (C) Nuclear (N) and cytoplasmic (C) extracts were immunoblotted with the indicated antibodies. (D) Sorted LT-HSCs were stained with antibodies against the indicated proteins and counterstained with PI for nucleus, followed by examination with confocal microscopy. (E) WASH is required for association of Rbbp4 with actin. Sorted LT-HSCs (2 × 10 5 ) from Ctrl or KO mice prepared as described above were immunoprecipitated with anti-Rbbp4 antibody and probed with the indicated antibodies. Antibodies against p16 and Atg5 served as positive and negative controls, respectively. IP, immunoprecipitation. (F) Sorted LT-HSCs (2 × 10 5 ) from WT mice cultured on stromal cells for 18 h were lysed for immunoprecipitation with antibody against WASH. Components of the NURF complex were blotted with the indicated antibodies. (G) Sorted LT-HSCs cultured with or without cytokines were lysed for ChIP assays with antibodies against Rbbp4, BPTF, or SNF2L. DNA levels were normalized to 5% input. (H) Nuclei were isolated from Ctrl or KO LT-HSCs with or without treatment of cytochalasin D for 6 h and digested by 1 unit of DNase I for the indicated times. DNA was then extracted for PCR analysis. (I and J) The VCA domain of WASH is required for c-Myc transcriptional activation. Sorted LT-HSCs from WASH KO mice were first infected with lentivirus encoding WT WASH or truncated mutant (WASH(ΔVCA)) in vitro and then transplanted into lethally irradiated CD45.1 mice together with 3 × 10 5 CD45.1 BM cells. 16 wk later, repopulation of BM cells were analyzed by flow cytometry for percentage of CD45.2 cells and numbers of LT-HSCs (I) and the mRNA levels of c-Myc in CD45.2 LT-HSCs were quantified by qRT-PCR analysis. mRNA levels were normalized to WT WASH rescued cells (J). Ctrl, n = 12; KO, n = 16. Bars, 2 µm. Data are shown as means ± SD. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: WASH is required for the differentiation commitment of hematopoietic stem cells in a c-Myc–dependent manner

    doi: 10.1084/jem.20140169

    Figure Lengend Snippet: WASH-mediated nuclear actin nucleation is needed to assist NURF complex to c-Myc promoter. (A and B) Nuclear and cytoplasmic extracts prepared from sorted LT-HSCs were incubated with recombinant WASH (rWASH; A) or nuclear extracts were incubated with various WASH mutants (B), followed by pyrene actin assays. (C) Nuclear (N) and cytoplasmic (C) extracts were immunoblotted with the indicated antibodies. (D) Sorted LT-HSCs were stained with antibodies against the indicated proteins and counterstained with PI for nucleus, followed by examination with confocal microscopy. (E) WASH is required for association of Rbbp4 with actin. Sorted LT-HSCs (2 × 10 5 ) from Ctrl or KO mice prepared as described above were immunoprecipitated with anti-Rbbp4 antibody and probed with the indicated antibodies. Antibodies against p16 and Atg5 served as positive and negative controls, respectively. IP, immunoprecipitation. (F) Sorted LT-HSCs (2 × 10 5 ) from WT mice cultured on stromal cells for 18 h were lysed for immunoprecipitation with antibody against WASH. Components of the NURF complex were blotted with the indicated antibodies. (G) Sorted LT-HSCs cultured with or without cytokines were lysed for ChIP assays with antibodies against Rbbp4, BPTF, or SNF2L. DNA levels were normalized to 5% input. (H) Nuclei were isolated from Ctrl or KO LT-HSCs with or without treatment of cytochalasin D for 6 h and digested by 1 unit of DNase I for the indicated times. DNA was then extracted for PCR analysis. (I and J) The VCA domain of WASH is required for c-Myc transcriptional activation. Sorted LT-HSCs from WASH KO mice were first infected with lentivirus encoding WT WASH or truncated mutant (WASH(ΔVCA)) in vitro and then transplanted into lethally irradiated CD45.1 mice together with 3 × 10 5 CD45.1 BM cells. 16 wk later, repopulation of BM cells were analyzed by flow cytometry for percentage of CD45.2 cells and numbers of LT-HSCs (I) and the mRNA levels of c-Myc in CD45.2 LT-HSCs were quantified by qRT-PCR analysis. mRNA levels were normalized to WT WASH rescued cells (J). Ctrl, n = 12; KO, n = 16. Bars, 2 µm. Data are shown as means ± SD. ***, P

    Article Snippet: Cytochalasin D (C6762) and Bafilomycin A1 (B1793) were from Sigma-Aldrich.

    Techniques: Incubation, Recombinant, Staining, Confocal Microscopy, Mouse Assay, Immunoprecipitation, Cell Culture, Chromatin Immunoprecipitation, Isolation, Polymerase Chain Reaction, Activation Assay, Infection, Mutagenesis, In Vitro, Irradiation, Flow Cytometry, Cytometry, Quantitative RT-PCR

    Disruption of cytoskeletal proteins abrogate nanofibrillar collagen-mediated EC alignment and elongation The addition of cytochalasin D (cyto D) or nocodazole (noco) inhibited F-actin assembly on aligned collagen substrates, based on immunofluorescence staining of F-actin or microtubules (A). Quantification of the angle of orientation (B) and cell shape index (C) in the presence of cytoskeleton disruption agents. Arrow denotes orientation of nanofibrils. Scale bar: 100 μm (A). **P

    Journal: Biomaterials

    Article Title: Spatial patterning of endothelium modulates cell morphology, adhesiveness and transcriptional signature

    doi: 10.1016/j.biomaterials.2013.01.017

    Figure Lengend Snippet: Disruption of cytoskeletal proteins abrogate nanofibrillar collagen-mediated EC alignment and elongation The addition of cytochalasin D (cyto D) or nocodazole (noco) inhibited F-actin assembly on aligned collagen substrates, based on immunofluorescence staining of F-actin or microtubules (A). Quantification of the angle of orientation (B) and cell shape index (C) in the presence of cytoskeleton disruption agents. Arrow denotes orientation of nanofibrils. Scale bar: 100 μm (A). **P

    Article Snippet: Immediately after plating the ECs on the collagen substrates, the ECs were incubated for 24 hours with cytoskeletal inhibitors cytochalasin D (100 nM, Sigma) or nocodazole (1 μM, Sigma), which disrupt the actin and microtubule networks, respectively (n=3).

    Techniques: Immunofluorescence, Staining

    B. pseudomallei is killed by NET formation. (A) Purified PMNs pretreated with medium alone, 100 nM the mitogen PMA, 10 μg/ml of the phagocytosis inhibitor cytochalasin D (Cyt D), or 100 units/ml of DNase I after incubation for 90 min from control

    Journal: Infection and Immunity

    Article Title: Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

    doi: 10.1128/IAI.00806-12

    Figure Lengend Snippet: B. pseudomallei is killed by NET formation. (A) Purified PMNs pretreated with medium alone, 100 nM the mitogen PMA, 10 μg/ml of the phagocytosis inhibitor cytochalasin D (Cyt D), or 100 units/ml of DNase I after incubation for 90 min from control

    Article Snippet: Purified PMNs were placed in 24-well tissue culture plates and incubated for 30 min at 37°C in the presence and absence of the phagocytosis inhibitor cytochalasin D (10 μg/ml; Sigma).

    Techniques: Purification, Incubation

    Neither apoptosis nor necroptosis is triggered by Candidalysin. a Phosphatidylserine exposure and cell viability of hMDMs infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strain ( ece1 Δ/Δ) (MOI 10) or treated with synthetic Candidalysin for 5 h were quantified by staining with FITC-Annexin V and PI, respectively. The number of single-stained or double-stained macrophages was evaluated by manual counting of at least 200 macrophages. b Caspase 3/7 activity was assessed by measuring luminescence of hMDMs 7 h post infection with C . albicans Wt or ece1 Δ/Δ mutant strain (MOI 10) or co-incubation with Candidalysin. Staurosporine served as a positive control. Shown are relative luminescence values (RLU) after background subtraction. c , d LPS-primed hMDMs were treated with synthetic Candidalysin or Nigericin for 5 h. Selected samples were pre-treated with c the necroptosis inhibitor Necrostatin-1 (Nec-1) or d the actin cytoskeleton inhibitor Cytochalasin D or inhibitor solute control DMSO 1 h prior to administration of synthetic Candidalysin or Nigericin. Macrophage lysis was quantified by measuring LDH release. e LPS-primed hMDMs, mBMDMs or mBMDCs were treated with synthetic Candidalysin or Nigericin for 4–5 h. Selected samples were pre-treated with the potassium channel inhibitor glibenclamide or inhibitor solute control DMSO 1 h prior to administration of synthetic Candidalysin or Nigericin. KCl was added after LPS priming. Macrophage lysis was quantified by measuring LDH release. a Data are shown as mean + SD of two different donors. b – e Values are represented as scatterplot with median of three independent donors or experiments ( n ≥ 3). For statistical analysis, a one-way ANOVA with Dunnett’s multiple comparison test was used. *** p ≤ 0.001, significance compared to Candidalysin treatment

    Journal: Nature Communications

    Article Title: The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes

    doi: 10.1038/s41467-018-06607-1

    Figure Lengend Snippet: Neither apoptosis nor necroptosis is triggered by Candidalysin. a Phosphatidylserine exposure and cell viability of hMDMs infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strain ( ece1 Δ/Δ) (MOI 10) or treated with synthetic Candidalysin for 5 h were quantified by staining with FITC-Annexin V and PI, respectively. The number of single-stained or double-stained macrophages was evaluated by manual counting of at least 200 macrophages. b Caspase 3/7 activity was assessed by measuring luminescence of hMDMs 7 h post infection with C . albicans Wt or ece1 Δ/Δ mutant strain (MOI 10) or co-incubation with Candidalysin. Staurosporine served as a positive control. Shown are relative luminescence values (RLU) after background subtraction. c , d LPS-primed hMDMs were treated with synthetic Candidalysin or Nigericin for 5 h. Selected samples were pre-treated with c the necroptosis inhibitor Necrostatin-1 (Nec-1) or d the actin cytoskeleton inhibitor Cytochalasin D or inhibitor solute control DMSO 1 h prior to administration of synthetic Candidalysin or Nigericin. Macrophage lysis was quantified by measuring LDH release. e LPS-primed hMDMs, mBMDMs or mBMDCs were treated with synthetic Candidalysin or Nigericin for 4–5 h. Selected samples were pre-treated with the potassium channel inhibitor glibenclamide or inhibitor solute control DMSO 1 h prior to administration of synthetic Candidalysin or Nigericin. KCl was added after LPS priming. Macrophage lysis was quantified by measuring LDH release. a Data are shown as mean + SD of two different donors. b – e Values are represented as scatterplot with median of three independent donors or experiments ( n ≥ 3). For statistical analysis, a one-way ANOVA with Dunnett’s multiple comparison test was used. *** p ≤ 0.001, significance compared to Candidalysin treatment

    Article Snippet: For inhibitor studies, the following compounds were added 1 h prior to infection: the caspase-1-inhibitor Z-YVAD-FMK (88.9 µM; Merck) or Ac-YVAD-cmk (20 µM, Invivogen), the caspase-1-inhibitor VX-765 (50 µg/mL, Invivogen) or vehicle control, the actin cytoskeleton inhibitor Cytochalasin D (10 µM; Sigma Aldrich), the V-ATPase inhibitor Bafilomycin A1 (50–500 nM; Sigma Aldrich), the ROS inhibitor 4-Aminopyrrolidine-2,4-dicarboxylate (PDTC) (100, 500 µM; Enzo Life Sciences), the potassium channel inhibitor glibenclamide (25 µM; Sigma Aldrich) or the RIP1-kinase inhibitor Necrostatin-1 (12.5–50 µM; Biomol).

    Techniques: Infection, Mutagenesis, Staining, Activity Assay, Incubation, Positive Control, Lysis

    Potassium-dependent and actin-dependent inflammasome activation. a IL-1β and IL-8 levels measured by ELISA in culture supernatants of LPS-primed hMDMs treated with synthetic Candidalysin or Nigericin for 5 h. Selected samples were pre-treated with the actin cytoskeleton inhibitor Cytochalasin D or the inhibitor solute control DMSO 1 h prior to administration of Candidalysin. b IL-1β release measured by ELISA in culture supernatants of LPS-primed hMDMs that were infected with C . albicans Wt, ece1 Δ/Δ, efg1 Δ/Δ/ cph1 Δ/Δ, hgc1 Δ/Δ mutant strains, or heat-killed Wt (MOI 10) in presence or absence of synthetic Candidalysin for 5 h. c – f IL-1β and c IL-8 or d , e TNF levels measured by ELISA in culture supernatants of LPS-primed c , f hMDMs d mBMDMs, and e mBMDCs. Phagocytes were treated with synthetic Candidalysin or Nigericin for 5 or 4 h (BMDCs). Selected samples were pre-treated with the following inhibitors 1 h prior to administration of Candidalysin: c – e the potassium channel inhibitor glibenclamide or inhibitor solute control DMSO, KCl was added after LPS priming, f (2R,4 R)-4-aminopyrrolidine-2,4-dicarboxylate (PDTC). g Intracellular ROS production in hMDMs pre-loaded with 20 μM H2DCF-DA for 30 min and infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 10) or treated with H 2 O 2 (positive control) for 5 h. Fluorescence (Ex 485/Em 535) measured immediately after infection was subtracted from fluorescence (Ex 485/Em 535) measured after 5 h. h Total ROS production in hMDMs subjected to synthetic Candidalysin or PMA (positive control) was monitored by Luminol-enhanced chemiluminescence. Relative luminescence units (RLU) were recorded for 60 min and the difference between maximum and minimum luminescence values was calculated. Data are represented as scatterplot and median of at least three different donors ( n ≥ 3) or independent experiments. For statistical analysis, a one-way ANOVA with Dunnett’s multiple comparison test was used. For analysis of the different C . albicans mutants, a two-way ANOVA with Sidak’s multiple comparison test was applied. *** p ≤ 0.001, * p ≤ 0.05, n/a not applicable, nd not detectable

    Journal: Nature Communications

    Article Title: The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes

    doi: 10.1038/s41467-018-06607-1

    Figure Lengend Snippet: Potassium-dependent and actin-dependent inflammasome activation. a IL-1β and IL-8 levels measured by ELISA in culture supernatants of LPS-primed hMDMs treated with synthetic Candidalysin or Nigericin for 5 h. Selected samples were pre-treated with the actin cytoskeleton inhibitor Cytochalasin D or the inhibitor solute control DMSO 1 h prior to administration of Candidalysin. b IL-1β release measured by ELISA in culture supernatants of LPS-primed hMDMs that were infected with C . albicans Wt, ece1 Δ/Δ, efg1 Δ/Δ/ cph1 Δ/Δ, hgc1 Δ/Δ mutant strains, or heat-killed Wt (MOI 10) in presence or absence of synthetic Candidalysin for 5 h. c – f IL-1β and c IL-8 or d , e TNF levels measured by ELISA in culture supernatants of LPS-primed c , f hMDMs d mBMDMs, and e mBMDCs. Phagocytes were treated with synthetic Candidalysin or Nigericin for 5 or 4 h (BMDCs). Selected samples were pre-treated with the following inhibitors 1 h prior to administration of Candidalysin: c – e the potassium channel inhibitor glibenclamide or inhibitor solute control DMSO, KCl was added after LPS priming, f (2R,4 R)-4-aminopyrrolidine-2,4-dicarboxylate (PDTC). g Intracellular ROS production in hMDMs pre-loaded with 20 μM H2DCF-DA for 30 min and infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 10) or treated with H 2 O 2 (positive control) for 5 h. Fluorescence (Ex 485/Em 535) measured immediately after infection was subtracted from fluorescence (Ex 485/Em 535) measured after 5 h. h Total ROS production in hMDMs subjected to synthetic Candidalysin or PMA (positive control) was monitored by Luminol-enhanced chemiluminescence. Relative luminescence units (RLU) were recorded for 60 min and the difference between maximum and minimum luminescence values was calculated. Data are represented as scatterplot and median of at least three different donors ( n ≥ 3) or independent experiments. For statistical analysis, a one-way ANOVA with Dunnett’s multiple comparison test was used. For analysis of the different C . albicans mutants, a two-way ANOVA with Sidak’s multiple comparison test was applied. *** p ≤ 0.001, * p ≤ 0.05, n/a not applicable, nd not detectable

    Article Snippet: For inhibitor studies, the following compounds were added 1 h prior to infection: the caspase-1-inhibitor Z-YVAD-FMK (88.9 µM; Merck) or Ac-YVAD-cmk (20 µM, Invivogen), the caspase-1-inhibitor VX-765 (50 µg/mL, Invivogen) or vehicle control, the actin cytoskeleton inhibitor Cytochalasin D (10 µM; Sigma Aldrich), the V-ATPase inhibitor Bafilomycin A1 (50–500 nM; Sigma Aldrich), the ROS inhibitor 4-Aminopyrrolidine-2,4-dicarboxylate (PDTC) (100, 500 µM; Enzo Life Sciences), the potassium channel inhibitor glibenclamide (25 µM; Sigma Aldrich) or the RIP1-kinase inhibitor Necrostatin-1 (12.5–50 µM; Biomol).

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Infection, Mutagenesis, Positive Control, Fluorescence

    Poly(I:C) and TLR3 colocalize at the surface of plasma membrane. HEK293T cells were transfected with TLR3-mCer alone (A) or cotransfected with UNC93B1 (B). Cells were stimulated with rhodamine labeled poly(I:C) (poly(I:C)-R). (B) TLR3 and poly(I:C)-R colocalization was evaluated from plots (right) of fluorescence intensities within 3 μm line profiles (n = 3; i, ii, iii). Three representative speckles where cross-sections were analyzed are marked with the white arrows on the merged image. (C–E) HEK293 cells were transfected with TLR3 alone or with UNC93B1 encoding plasmid. Cells were cotransfected with IFN-β (left) or NF-κB (right) promoter reporter plasmid and Renilla reporter plasmid. Cells were simultaneously treated with poly(I:C) (10 μg/ml) and inhibitors. Cells were treated with increasing amounts (0.2–5 μM) of cytochalasin D (abbr. CD) (C), Dynasore (abbr. Dy) (20–80 μM) (D) or bafilomycin A (abbr. BA) (0.2–2 μM) (E). 8 h after treatment luciferase activity (RLU) was measured in the cell lysates. The results are represented by mean values with SD from triplicate wells. The representative data from three experiments are shown.

    Journal: PLoS ONE

    Article Title: The Ectodomain of TLR3 Receptor Is Required for Its Plasma Membrane Translocation

    doi: 10.1371/journal.pone.0092391

    Figure Lengend Snippet: Poly(I:C) and TLR3 colocalize at the surface of plasma membrane. HEK293T cells were transfected with TLR3-mCer alone (A) or cotransfected with UNC93B1 (B). Cells were stimulated with rhodamine labeled poly(I:C) (poly(I:C)-R). (B) TLR3 and poly(I:C)-R colocalization was evaluated from plots (right) of fluorescence intensities within 3 μm line profiles (n = 3; i, ii, iii). Three representative speckles where cross-sections were analyzed are marked with the white arrows on the merged image. (C–E) HEK293 cells were transfected with TLR3 alone or with UNC93B1 encoding plasmid. Cells were cotransfected with IFN-β (left) or NF-κB (right) promoter reporter plasmid and Renilla reporter plasmid. Cells were simultaneously treated with poly(I:C) (10 μg/ml) and inhibitors. Cells were treated with increasing amounts (0.2–5 μM) of cytochalasin D (abbr. CD) (C), Dynasore (abbr. Dy) (20–80 μM) (D) or bafilomycin A (abbr. BA) (0.2–2 μM) (E). 8 h after treatment luciferase activity (RLU) was measured in the cell lysates. The results are represented by mean values with SD from triplicate wells. The representative data from three experiments are shown.

    Article Snippet: Cells were treated with different inhibitors: inhibitor of actin polymerization - cytochalasin D (Sigma Aldrich), inhibitor of dynamin – Dynasore (Sigma Aldrich) and inhibitor of endosomal acidification - bafilomycin A (LC Laboratories).

    Techniques: Transfection, Labeling, Fluorescence, Plasmid Preparation, Luciferase, Activity Assay

    Inhibition of bacterial uptake blocks PGE 2 biosynthesis by inoculated macrophages. (a) BMDMs were pretreated with 10 μM cytochalasin D (Cyto D) and then inoculated with F. tularensis LVS at an MOI of 200:1, and PGE 2 in the supernatant was quantified by enzyme-linked immunosorbent assay 24 hours after inoculation ( n = 3). (b and c) BMDMs (b) or RAW 264.7 macrophages (c) pretreated with inhibitors or vehicle and then inoculated with F. tularensis at an MOI of 200:1. At 6 and 24 hours after inoculation, macrophages were lysed and intramacrophage bacteria were quantified ( n = 3). The asterisk denotes a statistical difference compared to the untreated F. tularensis -infected group ( P ≤ 0.05). Graphs are means of pooled experiments ± SEMs.

    Journal: Infection and Immunity

    Article Title: Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases

    doi: 10.1128/IAI.02060-14

    Figure Lengend Snippet: Inhibition of bacterial uptake blocks PGE 2 biosynthesis by inoculated macrophages. (a) BMDMs were pretreated with 10 μM cytochalasin D (Cyto D) and then inoculated with F. tularensis LVS at an MOI of 200:1, and PGE 2 in the supernatant was quantified by enzyme-linked immunosorbent assay 24 hours after inoculation ( n = 3). (b and c) BMDMs (b) or RAW 264.7 macrophages (c) pretreated with inhibitors or vehicle and then inoculated with F. tularensis at an MOI of 200:1. At 6 and 24 hours after inoculation, macrophages were lysed and intramacrophage bacteria were quantified ( n = 3). The asterisk denotes a statistical difference compared to the untreated F. tularensis -infected group ( P ≤ 0.05). Graphs are means of pooled experiments ± SEMs.

    Article Snippet: The inhibitors used in these studies were pyrrophenone (for cPLA2 ), CAY10590 (for sPLA2 ), Edelfosine (for phosphatidylinositol [PI]-PLC), JZL184 (for MAGL), and indomethacin (for COX-2) (all purchased from Cayman Chemical); propranolol (for PAP), RHC80267 (for DAGL), MJ33 (for iPLA2 ), (for PLC), and cytochalasin D (CD) (for actin polymerization) (all purchased from Sigma-Aldrich); VU0155056 (for PLD1/2) (purchased from Avanti Polar Lipids, Inc.); and D609 (for phosphatidylcholine [PC]-PLC) (purchased from Tocris Bioscience).

    Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Infection

    S. enterica serovar Typhimurium induces the formation of actin-dependent membrane protrusions during basolateral invasion of polarized MDCK cells. (A and B) MDCK cells seeded on 3-μm-pore-size filter supports were infected from the basolateral pole with Salmonella and then fixed and processed for transmission electron microscopy (A) or confocal fluorescence microscopy (B). The red areas in panel B represent bacteria, and the green areas represent filamentous actin. The arrowhead in panel B indicates the region magnified in the inset. Bars: 1 μm (A), 10 μm (B). (C) MDCK cells were treated with 10 μg of cytochalasin D (CD)/ml and then subjected to a gentamicin resistance invasion assay. White bars, untreated cells; black bars, cells treated with CD. Results are expressed as mean invasion percentages ± standard deviations. *, P

    Journal: Infection and Immunity

    Article Title: Coordinate Regulation of Salmonella enterica Serovar Typhimurium Invasion of Epithelial Cells by the Arp2/3 Complex and Rho GTPases

    doi: 10.1128/IAI.71.5.2885-2891.2003

    Figure Lengend Snippet: S. enterica serovar Typhimurium induces the formation of actin-dependent membrane protrusions during basolateral invasion of polarized MDCK cells. (A and B) MDCK cells seeded on 3-μm-pore-size filter supports were infected from the basolateral pole with Salmonella and then fixed and processed for transmission electron microscopy (A) or confocal fluorescence microscopy (B). The red areas in panel B represent bacteria, and the green areas represent filamentous actin. The arrowhead in panel B indicates the region magnified in the inset. Bars: 1 μm (A), 10 μm (B). (C) MDCK cells were treated with 10 μg of cytochalasin D (CD)/ml and then subjected to a gentamicin resistance invasion assay. White bars, untreated cells; black bars, cells treated with CD. Results are expressed as mean invasion percentages ± standard deviations. *, P

    Article Snippet: MDCK cells cultured to confluency on filter supports were treated with the actin-depolymerizing agent cytochalasin D (Sigma Chemical Co., St. Louis, Mo.) at 10 μg/ml for 1 h prior to apical or basolateral infection with Salmonella , and the number of internalized bacteria was quantified using the gentamicin resistance assay as described previously ( ).

    Techniques: Infection, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy, Invasion Assay

    (A) Effects of inhibitors on EHEC- or EPEC-induced cortactin accumulation observed by confocal laser scanning microscopy. HeLa cells were treated with Cyt-D, staurosporine (STAU), or the drug PP1 (see the text for details) and were either not infected (CONTROL) or infected with EHEC O157:H7 or EPEC organisms. Cortactin (pink) was stained with anticortactin serum labeled with appropriate rhodamine-conjugated goat anti-mouse antibodies. The arrows indicate a bacterial cluster and the corresponding accumulation of cortactin at the bacterial adherence site. (B) Confocal images of cortactin accumulation by S. flexneri . HeLa cells were infected with S. flexneri for 30 min (A and B) or 1 h (C). Cortactin accumulation was observed in the absence (A and C) or presence (B) of PP1. The arrows show cortactin accumulation at the bacterial adherence site. See the text for details. Bar, 10 μm.

    Journal: Infection and Immunity

    Article Title: Interaction of Enteropathogenic or Enterohemorrhagic Escherichia coli with HeLa Cells Results in Translocation of Cortactin to the Bacterial Adherence Site

    doi:

    Figure Lengend Snippet: (A) Effects of inhibitors on EHEC- or EPEC-induced cortactin accumulation observed by confocal laser scanning microscopy. HeLa cells were treated with Cyt-D, staurosporine (STAU), or the drug PP1 (see the text for details) and were either not infected (CONTROL) or infected with EHEC O157:H7 or EPEC organisms. Cortactin (pink) was stained with anticortactin serum labeled with appropriate rhodamine-conjugated goat anti-mouse antibodies. The arrows indicate a bacterial cluster and the corresponding accumulation of cortactin at the bacterial adherence site. (B) Confocal images of cortactin accumulation by S. flexneri . HeLa cells were infected with S. flexneri for 30 min (A and B) or 1 h (C). Cortactin accumulation was observed in the absence (A and C) or presence (B) of PP1. The arrows show cortactin accumulation at the bacterial adherence site. See the text for details. Bar, 10 μm.

    Article Snippet: In some experiments, the inhibitors used, i.e., the F actin-depolymerizing agent cytochalasin D (Cyt-D; Sigma Chemical Co., St. Louis, Mo.) (2 μM) and the tyrosine protein kinase (TPK) inhibitor staurosporine (Sigma) (1 μM), were both added to HeLa cells 30 min prior to infection.

    Techniques: Confocal Laser Scanning Microscopy, Infection, Staining, Labeling

    Role of the RhoA/ROCK pathway in leptin-induced GATA-4 translocation. Representative confocal microscopic images of RASMC to study the effect of Y-27632 (Y; 10 μmol/L) or cytochalasin D (Cyto D; 1 μmol/L) on leptin-induced GATA-4 translocation. DAPI stained the nuclei (left panel; blue) and GATA-4 was detected using CruzFluor 488-conjugated secondary antibody (middle panel; green). The overlay (merged) is shown in the right panel. GATA-4 translocation in response to leptin was abolished by Y and Cyto D.

    Journal: Frontiers in Pharmacology

    Article Title: Mechanical stretch-induced vascular hypertrophy occurs through modulation of leptin synthesis-mediated ROS formation and GATA-4 nuclear translocation

    doi: 10.3389/fphar.2015.00240

    Figure Lengend Snippet: Role of the RhoA/ROCK pathway in leptin-induced GATA-4 translocation. Representative confocal microscopic images of RASMC to study the effect of Y-27632 (Y; 10 μmol/L) or cytochalasin D (Cyto D; 1 μmol/L) on leptin-induced GATA-4 translocation. DAPI stained the nuclei (left panel; blue) and GATA-4 was detected using CruzFluor 488-conjugated secondary antibody (middle panel; green). The overlay (merged) is shown in the right panel. GATA-4 translocation in response to leptin was abolished by Y and Cyto D.

    Article Snippet: Inhibitors such as the selective ROCK inhibitor Y-27632 (10 μmol/L, Sigma Aldrich, MO, USA), the actin depolymerization agent cytochalasin D (1 μmol/L; Calbiochem, CA, USA), the NADPH oxidase inhibitor apocynin (1 μmol/L, 4-Hydroxy-3-methoxyacetophenone, Sigma Aldrich, MO, USA), and the anti-leptin antibody Ob (Y-20) (1 μmol/L; Santa Cruz Biotechnology, CA, USA) were added to the media 60 min before mechanically stretching the RPV or adding leptin.

    Techniques: Translocation Assay, Staining

    Role of the RhoA/ROCK pathway in leptin- / mechanical stretch-induced ROS formation. Effect of Y-27632 (Y; 10 μmol/L) or cytochalasin D (Cyto D; 1 μmol/L) on mechanical stretch- (A) and leptin- (E) induced ROS formation. (C) . Effect of inhibition of leptin by anti-leptin antibody (Anti-Lep) on mechanical stretch-induced ROS formation. Fluorescence intensity measurements of DHE positive area of the mechanically stretched (B,D) and leptin-induced (F) RPVs treated with Anti-Lep, Y, or Cyto D ( n =3). * p

    Journal: Frontiers in Pharmacology

    Article Title: Mechanical stretch-induced vascular hypertrophy occurs through modulation of leptin synthesis-mediated ROS formation and GATA-4 nuclear translocation

    doi: 10.3389/fphar.2015.00240

    Figure Lengend Snippet: Role of the RhoA/ROCK pathway in leptin- / mechanical stretch-induced ROS formation. Effect of Y-27632 (Y; 10 μmol/L) or cytochalasin D (Cyto D; 1 μmol/L) on mechanical stretch- (A) and leptin- (E) induced ROS formation. (C) . Effect of inhibition of leptin by anti-leptin antibody (Anti-Lep) on mechanical stretch-induced ROS formation. Fluorescence intensity measurements of DHE positive area of the mechanically stretched (B,D) and leptin-induced (F) RPVs treated with Anti-Lep, Y, or Cyto D ( n =3). * p

    Article Snippet: Inhibitors such as the selective ROCK inhibitor Y-27632 (10 μmol/L, Sigma Aldrich, MO, USA), the actin depolymerization agent cytochalasin D (1 μmol/L; Calbiochem, CA, USA), the NADPH oxidase inhibitor apocynin (1 μmol/L, 4-Hydroxy-3-methoxyacetophenone, Sigma Aldrich, MO, USA), and the anti-leptin antibody Ob (Y-20) (1 μmol/L; Santa Cruz Biotechnology, CA, USA) were added to the media 60 min before mechanically stretching the RPV or adding leptin.

    Techniques: Inhibition, Fluorescence

    Role of the RhoA/ROCK pathway and intact cytoskeleton on Nox expression. Effect of Y-27632 (Y; 10 μmol/L) or cytochalasin D (Cyto D; 1 μmol/L) on mechanical stretch-induced Nox1 and Nox4 gene expression using qPCR analysis. ( n =8–10). * p

    Journal: Frontiers in Pharmacology

    Article Title: Mechanical stretch-induced vascular hypertrophy occurs through modulation of leptin synthesis-mediated ROS formation and GATA-4 nuclear translocation

    doi: 10.3389/fphar.2015.00240

    Figure Lengend Snippet: Role of the RhoA/ROCK pathway and intact cytoskeleton on Nox expression. Effect of Y-27632 (Y; 10 μmol/L) or cytochalasin D (Cyto D; 1 μmol/L) on mechanical stretch-induced Nox1 and Nox4 gene expression using qPCR analysis. ( n =8–10). * p

    Article Snippet: Inhibitors such as the selective ROCK inhibitor Y-27632 (10 μmol/L, Sigma Aldrich, MO, USA), the actin depolymerization agent cytochalasin D (1 μmol/L; Calbiochem, CA, USA), the NADPH oxidase inhibitor apocynin (1 μmol/L, 4-Hydroxy-3-methoxyacetophenone, Sigma Aldrich, MO, USA), and the anti-leptin antibody Ob (Y-20) (1 μmol/L; Santa Cruz Biotechnology, CA, USA) were added to the media 60 min before mechanically stretching the RPV or adding leptin.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Bacterial Omp29 up-regulates F-actin rearrangement in OBA9, which results in induction of Aa Y4 entry into OBA9. (A and B) Aa Y4 harvested at mid-log growth was incubated with confluent OBA9 cells (5×10 5 bacteria/100 µl/well) in the presence or absence of cytochalasin D (F-actin rearrangement inhibitor) by the indicated concentration in the figures. The number of bacteria entered or adherent to OBA9 cells was shown (A, bacterial entry; B; bacterial attachment). *, Significantly lower than control medium alone by Student's t test ( P

    Journal: PLoS ONE

    Article Title: Aggregatibacter actinomycetemcomitans Omp29 Is Associated with Bacterial Entry to Gingival Epithelial Cells by F-Actin Rearrangement

    doi: 10.1371/journal.pone.0018287

    Figure Lengend Snippet: Bacterial Omp29 up-regulates F-actin rearrangement in OBA9, which results in induction of Aa Y4 entry into OBA9. (A and B) Aa Y4 harvested at mid-log growth was incubated with confluent OBA9 cells (5×10 5 bacteria/100 µl/well) in the presence or absence of cytochalasin D (F-actin rearrangement inhibitor) by the indicated concentration in the figures. The number of bacteria entered or adherent to OBA9 cells was shown (A, bacterial entry; B; bacterial attachment). *, Significantly lower than control medium alone by Student's t test ( P

    Article Snippet: In some assays, phosphatidylinositol-3 (PI-3) kinase inhibitor Wortmannin (Biomol Research Laboratories, Plymouth Meeting, PA), a small GTP binding protein Rho inhibitor EDIN (epidermal cell differentiation inhibitor ), or actin rearrangement inhibitor cytochalasin D (Sigma, St. Louis, MO) was added to the medium.

    Techniques: Incubation, Concentration Assay

    Pharmacologic inhibition of cell tension reduces stiffness-dependent differences in cytoskeletal and adhesive architecture U87-MG cells cultured on fibronectin-coated glass and polyacrylamide substrates were treated with 25 μM blebbistatin, 10 μM Y-27632, or 1 μM cytochalasin D for 12–24 hours before fixation and staining for nuclear DNA (blue), F-actin (green), and vinculin (red). In all cases, the number and size of vinculin-positive focal adhesions was reduced with inhibition of cell tension. Blebbistatin and Y-27632 both induced cell spreading on the softest substrates, whereas cytochalasin D induced collapse of the actin cytoskeleton. Bar is 25 μm.

    Journal: Cancer research

    Article Title: The mechanical rigidity of the extracellular matrix regulates the structure, motility, and proliferation of glioma cells

    doi: 10.1158/0008-5472.CAN-08-4859

    Figure Lengend Snippet: Pharmacologic inhibition of cell tension reduces stiffness-dependent differences in cytoskeletal and adhesive architecture U87-MG cells cultured on fibronectin-coated glass and polyacrylamide substrates were treated with 25 μM blebbistatin, 10 μM Y-27632, or 1 μM cytochalasin D for 12–24 hours before fixation and staining for nuclear DNA (blue), F-actin (green), and vinculin (red). In all cases, the number and size of vinculin-positive focal adhesions was reduced with inhibition of cell tension. Blebbistatin and Y-27632 both induced cell spreading on the softest substrates, whereas cytochalasin D induced collapse of the actin cytoskeleton. Bar is 25 μm.

    Article Snippet: Rho-associated kinase (ROCK) inhibitor Y-27632 (Calbiochem, La Jolla, CA), NMMII inhibitor blebbistatin (Sigma-Aldrich), and actin polymerization inhibitor cytochalasin D (Sigma-Aldrich) were added to the cell culture media in relevant timelapse and immunofluorescence experiments after the cells had been allowed to adhere for at least 10 hours.

    Techniques: Inhibition, Cell Culture, Staining

    Pharmacologic inhibition of cytoskeletal contractility reduces stiffness-dependent differences in cell morphology (A) U87-MG cells cultured on fibronectin-conjugated glass and polyacrylamide substrates in either the absence of drug (control) or 24 hours after addition of 25 μM blebbistatin, 10 μM Y-27632, or 1 μM cytochalasin D. Cells began extending actin-rich processes (arrows) within an hour after addition of Y-27632 or blebbistatin. Cytochalasin D induced stellation and rounding of cells on stiff substrates but had no appreciable effect on the morphology or migration of cells on the softest substrates. Bar is 100 μm. (B) U373-MG cells cultured on 0.08 kPa fibronectin-conjugated polyacrylamide substrates showed enhanced spreading and migration with addition of 50 μM Y-27632. Bar is 100 μm.

    Journal: Cancer research

    Article Title: The mechanical rigidity of the extracellular matrix regulates the structure, motility, and proliferation of glioma cells

    doi: 10.1158/0008-5472.CAN-08-4859

    Figure Lengend Snippet: Pharmacologic inhibition of cytoskeletal contractility reduces stiffness-dependent differences in cell morphology (A) U87-MG cells cultured on fibronectin-conjugated glass and polyacrylamide substrates in either the absence of drug (control) or 24 hours after addition of 25 μM blebbistatin, 10 μM Y-27632, or 1 μM cytochalasin D. Cells began extending actin-rich processes (arrows) within an hour after addition of Y-27632 or blebbistatin. Cytochalasin D induced stellation and rounding of cells on stiff substrates but had no appreciable effect on the morphology or migration of cells on the softest substrates. Bar is 100 μm. (B) U373-MG cells cultured on 0.08 kPa fibronectin-conjugated polyacrylamide substrates showed enhanced spreading and migration with addition of 50 μM Y-27632. Bar is 100 μm.

    Article Snippet: Rho-associated kinase (ROCK) inhibitor Y-27632 (Calbiochem, La Jolla, CA), NMMII inhibitor blebbistatin (Sigma-Aldrich), and actin polymerization inhibitor cytochalasin D (Sigma-Aldrich) were added to the cell culture media in relevant timelapse and immunofluorescence experiments after the cells had been allowed to adhere for at least 10 hours.

    Techniques: Inhibition, Cell Culture, Migration

    Inflammasome priming by Francisella is independent of bacteria internalization. Human monocytes were pretreated for 30 min with latrunculin A (Latr) (200 nM) and cytochalasin D (Cyto D) (5 μg/ml) and infected with F . novicida for 16 h. CFU of internalized bacteria ( A ) and IL-18 release ( B ) were counted. Monocytes treated with latrunculin and cytochalasin D as in A, B and then primed with Francisella for 30 min followed by ATP (5mM) for 30 min were analyzed for IL-18 in cell culture media by ELISA ( C ). Data represent mean ± SEM, n = 3 independent experiments. * p

    Journal: PLoS ONE

    Article Title: Inflammasome Priming Is Similar for Francisella Species That Differentially Induce Inflammasome Activation

    doi: 10.1371/journal.pone.0127278

    Figure Lengend Snippet: Inflammasome priming by Francisella is independent of bacteria internalization. Human monocytes were pretreated for 30 min with latrunculin A (Latr) (200 nM) and cytochalasin D (Cyto D) (5 μg/ml) and infected with F . novicida for 16 h. CFU of internalized bacteria ( A ) and IL-18 release ( B ) were counted. Monocytes treated with latrunculin and cytochalasin D as in A, B and then primed with Francisella for 30 min followed by ATP (5mM) for 30 min were analyzed for IL-18 in cell culture media by ELISA ( C ). Data represent mean ± SEM, n = 3 independent experiments. * p

    Article Snippet: Phagocytosis was blocked by actin polymerization inhibitors cytochalasin D (Sigma-Aldrich, St. Louis, MO) and latrunculin (Cayman Chemical, Ann Arbor, MI).

    Techniques: Infection, Cell Culture, Enzyme-linked Immunosorbent Assay

    Effects of actin manipulation on oocyte effective tension. (A) Phalloidin staining of a GVI oocyte (left) and a metaphase II (MII) egg (right). Polarity develops during meiotic maturation, such that the metaphase II egg has an actin-rich cap over the meiotic spindle. Red, actin; blue, DNA. Scale bar, (left) 20 μm; (right) 22 μm. (B) Effects on effective tension (T eff ) of actin manipulation by cytochalasin D treatment of GVI oocytes (*p

    Journal: Molecular Biology of the Cell

    Article Title: Cortical Mechanics and Meiosis II Completion in Mammalian Oocytes Are Mediated by Myosin-II and Ezrin-Radixin-Moesin (ERM) Proteins

    doi: 10.1091/mbc.E10-01-0066

    Figure Lengend Snippet: Effects of actin manipulation on oocyte effective tension. (A) Phalloidin staining of a GVI oocyte (left) and a metaphase II (MII) egg (right). Polarity develops during meiotic maturation, such that the metaphase II egg has an actin-rich cap over the meiotic spindle. Red, actin; blue, DNA. Scale bar, (left) 20 μm; (right) 22 μm. (B) Effects on effective tension (T eff ) of actin manipulation by cytochalasin D treatment of GVI oocytes (*p

    Article Snippet: Treatment with the actin filament disruptor cytochalasin D (5 μg/ml; Calbiochem, Gibbstown, NJ) or the myosin light-chain kinase inhibitor ML-7 (15 μM; Calbiochem, La Jolla, CA; or Sigma-Aldrich) was done as previously described ( ; ) for 60 min before and during Teff measurement by MPA.

    Techniques: Staining

    Spindle defects upon exit from metaphase II arrest with actin, myosin-II, or ERM disruption. Eggs were inseminated for 1.5 h (A–P) or 4 h (Q-BB). Control fertilized eggs (A–C, J–M, and Q–T) show normal spindle rotation and second polar body morphology, as illustrated in the schematic diagram. A failure in spindle rotation was observed in eggs treated with the MLCK inhibitor ML-7 (D–F; 73/75 eggs) and in eggs treated with the actin filament disruptor cytochalasin D (G–I; 34/34 eggs). Distorted, curved spindles were observed in DN-RDX–expressing eggs (N–P, U-BB; 26/36 eggs). In these embryos, two polar body-like (PBL) structures formed; these PBL structures did not resolve with increased time after insemination (U-BB). Arrowheads identify polar bodies (PB) and PBL structures. Maternal DNA is labeled m, and sperm DNA is labeled s; some eggs are polyspermic, although this is not uncommon with the insemination conditions used, particularly with cytochalasin D–treated eggs ( McAvey et al. , 2002 ). In several panels, DNA of the fertilizing sperm is out of the plane of focus. FC (T) identifies the fertilization cone containing the DNA of a fertilizing sperm. Scale bar, (DD) 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Cortical Mechanics and Meiosis II Completion in Mammalian Oocytes Are Mediated by Myosin-II and Ezrin-Radixin-Moesin (ERM) Proteins

    doi: 10.1091/mbc.E10-01-0066

    Figure Lengend Snippet: Spindle defects upon exit from metaphase II arrest with actin, myosin-II, or ERM disruption. Eggs were inseminated for 1.5 h (A–P) or 4 h (Q-BB). Control fertilized eggs (A–C, J–M, and Q–T) show normal spindle rotation and second polar body morphology, as illustrated in the schematic diagram. A failure in spindle rotation was observed in eggs treated with the MLCK inhibitor ML-7 (D–F; 73/75 eggs) and in eggs treated with the actin filament disruptor cytochalasin D (G–I; 34/34 eggs). Distorted, curved spindles were observed in DN-RDX–expressing eggs (N–P, U-BB; 26/36 eggs). In these embryos, two polar body-like (PBL) structures formed; these PBL structures did not resolve with increased time after insemination (U-BB). Arrowheads identify polar bodies (PB) and PBL structures. Maternal DNA is labeled m, and sperm DNA is labeled s; some eggs are polyspermic, although this is not uncommon with the insemination conditions used, particularly with cytochalasin D–treated eggs ( McAvey et al. , 2002 ). In several panels, DNA of the fertilizing sperm is out of the plane of focus. FC (T) identifies the fertilization cone containing the DNA of a fertilizing sperm. Scale bar, (DD) 10 μm.

    Article Snippet: Treatment with the actin filament disruptor cytochalasin D (5 μg/ml; Calbiochem, Gibbstown, NJ) or the myosin light-chain kinase inhibitor ML-7 (15 μM; Calbiochem, La Jolla, CA; or Sigma-Aldrich) was done as previously described ( ; ) for 60 min before and during Teff measurement by MPA.

    Techniques: Expressing, Labeling

    Effects of cytochalasin D treatment on neural patterning of ECM treated-hiPSCs. The cells treated with ECMs and ± cyto D at day 16 were analyzed for forebrain and hindbrain markers. (A) Representative flow cytometry histograms of forebrain marker

    Journal: ACS biomaterials science & engineering

    Article Title: Differential Effects of Heparin and Hyaluronic Acid on Neural Patterning of Human Induced Pluripotent Stem Cells

    doi: 10.1021/acsbiomaterials.8b01142

    Figure Lengend Snippet: Effects of cytochalasin D treatment on neural patterning of ECM treated-hiPSCs. The cells treated with ECMs and ± cyto D at day 16 were analyzed for forebrain and hindbrain markers. (A) Representative flow cytometry histograms of forebrain marker

    Article Snippet: To evaluate the impact of Hippo/YAP signaling, the stress fiber disruptor Cytochalasin D (2 μ M, Sigma) was added to the replated cells together with the ECMs at day 16.

    Techniques: Flow Cytometry, Cytometry, Marker

    Random distribution of labeled DNA-strands among daughter nuclei of LRCs. Analysis of the distribution of labeled DNA-strands among daughter nuclei of label-retaining cells (LRCs) on single cell level, using the actin-binding protein cytochalasin D. Cytochalasin D is an actin-binding protein that inhibits cytokinesis, while karyokinesis remains unaffected. Thereby, binucleate cells are created, which enables analysis of the distribution of DNA-strands among daughter nuclei on single cell level. (A): Scheme of the experimental set-up. Animals were pulsed continuously with EdU during embryonic development (day1–day5) and during post-embryonic development (day6–day11), followed by a chase time of 2 months in EdU-free medium. Subsequently animals were soaked in cytochalasin D for 7 days, EdU was visualized and DNA was stained with DAPI. (B): Visualization of binucleate LRCs in macerated cell suspensions. Fluorescence images of EdU (left), DAPI (middle), and interference contrast images (right) of binucleate LRCs, pulsed during embryonic (B1) and post-embryonic (B2, B3) development. Binucleate EdU + cells display equivalent EdU-signal in both daughter nuclei. Abbreviations: EdU, 5-ethynyl-2′-deoxyuridine. Scale bars: 5 µm.

    Journal: PLoS ONE

    Article Title: Stem Cells Propagate Their DNA by Random Segregation in the Flatworm Macrostomum lignano

    doi: 10.1371/journal.pone.0030227

    Figure Lengend Snippet: Random distribution of labeled DNA-strands among daughter nuclei of LRCs. Analysis of the distribution of labeled DNA-strands among daughter nuclei of label-retaining cells (LRCs) on single cell level, using the actin-binding protein cytochalasin D. Cytochalasin D is an actin-binding protein that inhibits cytokinesis, while karyokinesis remains unaffected. Thereby, binucleate cells are created, which enables analysis of the distribution of DNA-strands among daughter nuclei on single cell level. (A): Scheme of the experimental set-up. Animals were pulsed continuously with EdU during embryonic development (day1–day5) and during post-embryonic development (day6–day11), followed by a chase time of 2 months in EdU-free medium. Subsequently animals were soaked in cytochalasin D for 7 days, EdU was visualized and DNA was stained with DAPI. (B): Visualization of binucleate LRCs in macerated cell suspensions. Fluorescence images of EdU (left), DAPI (middle), and interference contrast images (right) of binucleate LRCs, pulsed during embryonic (B1) and post-embryonic (B2, B3) development. Binucleate EdU + cells display equivalent EdU-signal in both daughter nuclei. Abbreviations: EdU, 5-ethynyl-2′-deoxyuridine. Scale bars: 5 µm.

    Article Snippet: Animals were then chased in the presence of food (ad libitum ) for two months in EdU-free medium, followed by a seven-day incubation period in the actin-binding protein cytochalasin D (5 µM in f/2 - Sigma).

    Techniques: Labeling, Binding Assay, Staining, Fluorescence

    Eukaryotic cytoskeleton is important for  K. pneumoniae  invasion. Invasion assays of  K. pneumoniae  Ca0438 using Caco-2 cells were performed in the presence of compounds that interfered with host actin (cytochalasin D) or microtubules (nocodazole). Mock invasion in the absence of inhibitors was defined as 100% relative invasiveness. Data are presented as means ± SEM. *,  P

    Journal: Infection and Immunity

    Article Title: Klebsiella pneumoniae Translocates across the Intestinal Epithelium via Rho GTPase- and Phosphatidylinositol 3-Kinase/Akt-Dependent Cell Invasion

    doi: 10.1128/IAI.02345-14

    Figure Lengend Snippet: Eukaryotic cytoskeleton is important for K. pneumoniae invasion. Invasion assays of K. pneumoniae Ca0438 using Caco-2 cells were performed in the presence of compounds that interfered with host actin (cytochalasin D) or microtubules (nocodazole). Mock invasion in the absence of inhibitors was defined as 100% relative invasiveness. Data are presented as means ± SEM. *, P

    Article Snippet: The inhibitors that were analyzed were related to eukaryotic cytoskeleton dynamics or signal transduction: cytochalasin D (Sigma-Aldrich), nocodazole (Sigma-Aldrich), ML141 (Merck Millipore, Darmstadt, Germany), (Merck Millipore), PP1 (Merck Millipore), genistein (Merck Millipore); Rho inhibitor I (Cytoskeleton, Denver, CO), Rac1 inhibitor (Merck Millipore), and Akt1/2 kinase inhibitor (Merck Millipore).

    Techniques:

    Human PMNs kill T . vaginalis . (A) Schematic for T . vaginalis cytotoxicity assay: T . vaginalis and PMNs were labelled with CT or CFSE, respectively, and cocultured for 2 hours. Cells were then analyzed by flow cytometry to assess the survival of T . vaginalis . (B) Representative FACS plots of cytotoxicity assay results using an MOI of 0.25 are shown. Surviving T . vaginalis were defined as CT + CFSE − . (C) Percent cytotoxicity of T . vaginalis was determined by counting the number of surviving T . vaginalis as outlined in A and B, at various MOIs, defined as parasite:host cell ratio. All data are represented as mean ± SD. (D) Composite results of T . vaginalis FCSfiles. CFSE, Carboxyfluorescein succinimidyl ester; CT, Cell Tracker; FACS, fluorescence-activated cell sorting; FCS, fluorescence correlation spectroscopy; MOI, multiplicity of infection; PMN, polymorphonuclear cell.

    Journal: PLoS Biology

    Article Title: Neutrophils kill the parasite Trichomonas vaginalis using trogocytosis

    doi: 10.1371/journal.pbio.2003885

    Figure Lengend Snippet: Human PMNs kill T . vaginalis . (A) Schematic for T . vaginalis cytotoxicity assay: T . vaginalis and PMNs were labelled with CT or CFSE, respectively, and cocultured for 2 hours. Cells were then analyzed by flow cytometry to assess the survival of T . vaginalis . (B) Representative FACS plots of cytotoxicity assay results using an MOI of 0.25 are shown. Surviving T . vaginalis were defined as CT + CFSE − . (C) Percent cytotoxicity of T . vaginalis was determined by counting the number of surviving T . vaginalis as outlined in A and B, at various MOIs, defined as parasite:host cell ratio. All data are represented as mean ± SD. (D) Composite results of T . vaginalis FCSfiles. CFSE, Carboxyfluorescein succinimidyl ester; CT, Cell Tracker; FACS, fluorescence-activated cell sorting; FCS, fluorescence correlation spectroscopy; MOI, multiplicity of infection; PMN, polymorphonuclear cell.

    Article Snippet: Where noted, inhibitors/activators were added to T . vaginalis cytotoxicity assays either at the time of coculture (DNase-1 [Worthington], Catalase [Sigma], PMA [Cayman Chemical]), to PMNs 10 minutes before addition of T . vaginalis (Human IgG F(c) fragment [Rockland Chemical]), or to PMNs 20 minutes before the addition of T . vaginalis (Cytochalasin D [Sigma], wortmannin [Tocris], AEBSF [Tocris]).

    Techniques: Cytotoxicity Assay, Flow Cytometry, Cytometry, FACS, Fluorescence, Spectroscopy, Infection

    PMNs swarm and trogocytose T . vaginalis prior to T . vaginalis death. (A) Live imaging. Total T . vaginalis surface proteins were covalently linked with Biotin, stained with Streptavidin-Alexa-488 (A488, green), and cocultured with unlabeled PMNs at MOI 0.1 with 10 ug/ml PI in the media. Selected time points of 2D live video at 63X magnification of the interactions are shown. Images are representative of 96 parasite death events performed with PMNs from 11 different human donors. min. = minutes after parasites and PMNs were cocultured. Scale bar = 10 um. (B) and (C) Box and whisker plots showing distribution according to quartiles. (B) 96 videos of T . vaginalis death events from 11 donors’ PMNs were analyzed for the number of PMNs in contact with T . vaginalis before the parasite dies. (C) 96 videos from 11 donors’ PMNs were analyzed for the number of “bites” of T . vaginalis material transferred to PMNs before the parasite dies. (D) Videos in which the first “bite” was observable (19 videos from 7 donors’ PMNs) were analyzed for the time elapsed from when the first bite was taken until PI signal was observed in T . vaginalis . (E) 3D live imaging. T . vaginalis surface was labelled with Alexa-488 as in (A) and cocultured with CT-labelled PMNs (magenta) at MOI 0.1 in the presence of 10 ug/ml PI. Z-stacks spanning 15 um were acquired every 1.5 seconds. Selected time points at 63x of PMN engulfment of T . vaginalis material before parasite death are shown with 3D reconstruction of deconvolved images. Images are clipped midway through the z-axis to visualize the inside of the PMNs. Scale bar = 5 um. White arrow indicates “bite” of T . vaginalis that is uptaken by PMNs. (F) Last timeframe of (E) is shown without any clipping of the z-axis. Data in E and F are representative of at least 3 replicates each from 3 donors and 3 independent experiments. BF, bright field; CT, Cell Tracker; MOI, multiplicity of infection; PI, Propidium Iodide; PMN, polymorphonuclear cell.

    Journal: PLoS Biology

    Article Title: Neutrophils kill the parasite Trichomonas vaginalis using trogocytosis

    doi: 10.1371/journal.pbio.2003885

    Figure Lengend Snippet: PMNs swarm and trogocytose T . vaginalis prior to T . vaginalis death. (A) Live imaging. Total T . vaginalis surface proteins were covalently linked with Biotin, stained with Streptavidin-Alexa-488 (A488, green), and cocultured with unlabeled PMNs at MOI 0.1 with 10 ug/ml PI in the media. Selected time points of 2D live video at 63X magnification of the interactions are shown. Images are representative of 96 parasite death events performed with PMNs from 11 different human donors. min. = minutes after parasites and PMNs were cocultured. Scale bar = 10 um. (B) and (C) Box and whisker plots showing distribution according to quartiles. (B) 96 videos of T . vaginalis death events from 11 donors’ PMNs were analyzed for the number of PMNs in contact with T . vaginalis before the parasite dies. (C) 96 videos from 11 donors’ PMNs were analyzed for the number of “bites” of T . vaginalis material transferred to PMNs before the parasite dies. (D) Videos in which the first “bite” was observable (19 videos from 7 donors’ PMNs) were analyzed for the time elapsed from when the first bite was taken until PI signal was observed in T . vaginalis . (E) 3D live imaging. T . vaginalis surface was labelled with Alexa-488 as in (A) and cocultured with CT-labelled PMNs (magenta) at MOI 0.1 in the presence of 10 ug/ml PI. Z-stacks spanning 15 um were acquired every 1.5 seconds. Selected time points at 63x of PMN engulfment of T . vaginalis material before parasite death are shown with 3D reconstruction of deconvolved images. Images are clipped midway through the z-axis to visualize the inside of the PMNs. Scale bar = 5 um. White arrow indicates “bite” of T . vaginalis that is uptaken by PMNs. (F) Last timeframe of (E) is shown without any clipping of the z-axis. Data in E and F are representative of at least 3 replicates each from 3 donors and 3 independent experiments. BF, bright field; CT, Cell Tracker; MOI, multiplicity of infection; PI, Propidium Iodide; PMN, polymorphonuclear cell.

    Article Snippet: Where noted, inhibitors/activators were added to T . vaginalis cytotoxicity assays either at the time of coculture (DNase-1 [Worthington], Catalase [Sigma], PMA [Cayman Chemical]), to PMNs 10 minutes before addition of T . vaginalis (Human IgG F(c) fragment [Rockland Chemical]), or to PMNs 20 minutes before the addition of T . vaginalis (Cytochalasin D [Sigma], wortmannin [Tocris], AEBSF [Tocris]).

    Techniques: Imaging, Staining, Whisker Assay, Infection

    HSV-1 infection of keratinocytes pretreated with inhibitors against microtubules, actin, clathrin, sodium/proton exchanger or with lysosomotropic agents. HaCaT cells or primary human keratinocytes were pretreated with the indicated concentrations of drugs or corresponding solvent controls (C) for 30 min followed by infection with HSV-1 in the presence of the drugs. All infections were at 20 PFU/cell apart from the nocodazole (noc)-treated cells which were infected both at 20 and 5 PFU/cell. All cells were fixed at 2 h p.i. and stained with mouse anti-ICP0. The number of ICP0-expressing cells was determined in at least three independent experiments. The location of ICP0 expression was recorded as only in the nucleus (nuclear ICP0) or in both nucleus and cytoplasm (cytoplasmic ICP0) representing early and early-late phases during infection, respectively. The percentages of infected cells are shown after treatment with nocodazole (noc) in panels A and B, with NH 4 Cl and monensin (mon) in C and D, and with chlorpromazine (CPZ), EIPA and cytochalasin D (CD) in E and F. Results are mean ± standard deviation values. P-values (pair-t-test) are given above the diagram (B, D).

    Journal: PLoS ONE

    Article Title: Entry Pathways of Herpes Simplex Virus Type 1 into Human Keratinocytes Are Dynamin- and Cholesterol-Dependent

    doi: 10.1371/journal.pone.0025464

    Figure Lengend Snippet: HSV-1 infection of keratinocytes pretreated with inhibitors against microtubules, actin, clathrin, sodium/proton exchanger or with lysosomotropic agents. HaCaT cells or primary human keratinocytes were pretreated with the indicated concentrations of drugs or corresponding solvent controls (C) for 30 min followed by infection with HSV-1 in the presence of the drugs. All infections were at 20 PFU/cell apart from the nocodazole (noc)-treated cells which were infected both at 20 and 5 PFU/cell. All cells were fixed at 2 h p.i. and stained with mouse anti-ICP0. The number of ICP0-expressing cells was determined in at least three independent experiments. The location of ICP0 expression was recorded as only in the nucleus (nuclear ICP0) or in both nucleus and cytoplasm (cytoplasmic ICP0) representing early and early-late phases during infection, respectively. The percentages of infected cells are shown after treatment with nocodazole (noc) in panels A and B, with NH 4 Cl and monensin (mon) in C and D, and with chlorpromazine (CPZ), EIPA and cytochalasin D (CD) in E and F. Results are mean ± standard deviation values. P-values (pair-t-test) are given above the diagram (B, D).

    Article Snippet: Inhibitor studies Cytochalasin D and nocodazole (Sigma), and the dynamin inhibitors dynasore (Tocris) and MiTMAB (Calbiochem) were dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Infection, Staining, Expressing, Standard Deviation