cytochalasin d  (Millipore)


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    Structured Review

    Millipore cytochalasin d
    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 <t>Cytochalasin</t> D treatment ( e ) or CK666 treatment ( f ). The error bars indicate 95% confidence interval of the mean of the cluster proportions. * p
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 322 article reviews
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    cytochalasin d - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04030-0

    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
    Figure Legend 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

    Techniques Used: Functional Assay

    2) Product Images from "STAT6/Arg1 promotes microglia/macrophage efferocytosis and inflammation resolution in stroke mice"

    Article Title: STAT6/Arg1 promotes microglia/macrophage efferocytosis and inflammation resolution in stroke mice

    Journal: JCI Insight

    doi: 10.1172/jci.insight.131355

    STAT6 deficiency negates microglia/macrophage-induced protection of ischemic neurons. ( A ) Experimental design. Primary neurons were subjected to 90-minute OGD and then cocultured with WT or STAT6-KO microglia/macrophages. Neuronal survival was measured 24 hours later by MAP2 staining. ( B ) Representative images of MAP2-stained neurons after coculturing with WT or STAT6-KO microglia. Scale bar: 50 μm. ( C and D ) The number of MAP2 + (red) neurons was quantified 24 hours after coculturing with WT or STAT6-KO microglia ( C ) or macrophages ( D ). In some groups, WT microglia or macrophages were treated with cytochalasin D (10 μM, 1 hour) to inhibit phagocytosis. Data represent 3 independent experiments in duplicate. ** P ≤ 0.01 vs. OGD alone (2nd bar), 1-way ANOVA.
    Figure Legend Snippet: STAT6 deficiency negates microglia/macrophage-induced protection of ischemic neurons. ( A ) Experimental design. Primary neurons were subjected to 90-minute OGD and then cocultured with WT or STAT6-KO microglia/macrophages. Neuronal survival was measured 24 hours later by MAP2 staining. ( B ) Representative images of MAP2-stained neurons after coculturing with WT or STAT6-KO microglia. Scale bar: 50 μm. ( C and D ) The number of MAP2 + (red) neurons was quantified 24 hours after coculturing with WT or STAT6-KO microglia ( C ) or macrophages ( D ). In some groups, WT microglia or macrophages were treated with cytochalasin D (10 μM, 1 hour) to inhibit phagocytosis. Data represent 3 independent experiments in duplicate. ** P ≤ 0.01 vs. OGD alone (2nd bar), 1-way ANOVA.

    Techniques Used: Staining

    3) Product Images from "Gene Disruption of Plasmodium falciparum p52 Results in Attenuation of Malaria Liver Stage Development in Cultured Primary Human Hepatocytes"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0003549

    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.
    Figure Legend 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.

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

    4) Product Images from "Responses to Cell Loss Become Restricted as the Supporting Cells in Mammalian Vestibular Organs Grow Thick Junctional Actin Bands That Develop High Stability"

    Article Title: Responses to Cell Loss Become Restricted as the Supporting Cells in Mammalian Vestibular Organs Grow Thick Junctional Actin Bands That Develop High Stability

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4355-13.2014

    LatA and CytoD rapidly and reversibly disassemble circumferential F-actin bands in MDCK epithelial cells. A–C , Confocal images of MDCK cells treated with vehicle, 5 μ m LatA, or 100 n m CytoD for 15 min (left), followed by washout and an
    Figure Legend Snippet: LatA and CytoD rapidly and reversibly disassemble circumferential F-actin bands in MDCK epithelial cells. A–C , Confocal images of MDCK cells treated with vehicle, 5 μ m LatA, or 100 n m CytoD for 15 min (left), followed by washout and an

    Techniques Used:

    CytoD thins the wide actin bands at the junctions of SCs in adult mice. A , Confocal images of adult mouse utricles that were cultured with 100 n m CytoD or vehicle alone for 1–24 h and then fixed and labeled with fluorescent phalloidin. B , Images
    Figure Legend Snippet: CytoD thins the wide actin bands at the junctions of SCs in adult mice. A , Confocal images of adult mouse utricles that were cultured with 100 n m CytoD or vehicle alone for 1–24 h and then fixed and labeled with fluorescent phalloidin. B , Images

    Techniques Used: Mouse Assay, Cell Culture, Labeling

    Treatment with CytoD causes the circumferential bands in utricles from newborn mice and young chickens to appear frayed, but reductions in their widths were minimal. A , Confocal images of the sensory epithelium (SE) in P0 mouse utricles that were cultured
    Figure Legend Snippet: Treatment with CytoD causes the circumferential bands in utricles from newborn mice and young chickens to appear frayed, but reductions in their widths were minimal. A , Confocal images of the sensory epithelium (SE) in P0 mouse utricles that were cultured

    Techniques Used: Mouse Assay, Cell Culture

    5) Product Images from "TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro‐Phagocytic Signals From Apoptotic to Viable Cells"

    Article Title: TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro‐Phagocytic Signals From Apoptotic to Viable Cells

    Journal: Journal of Cellular Physiology

    doi: 10.1002/jcp.25584

    Inhibition of TNT formation deceased phagocytosis of viable PC12 cells. (A) Dose‐dependent inhibition of TNT number in PC12 cells after 24 h treatment with cytoD. Scale bars, 20 μm. (B) PS‐positive TNTs in PC12 cells were abolished by 24 h treatment of 50 nM cytoD. Scale bars, 30 μm. (C) 50 nM cytoD did not inhibit phagocytosis of apoptotic PC12 cells. DiD‐RAW264.7 cells were cocultured with CTG‐stained untreated or UV‐treated PC12 cells in the absence and presence of 50 nM cytoD for 24 h, resp. Phagocytosis was measured by flow cytometry, and expressed as the percentage of CTG/DiD double‐positive RAW264.7 cells as a subpopulation of all DiD‐positive RAW264.7 cells. (D) Phagocytosis of viable PC12 cells was inhibited by 50 nM cytoD. CTG‐PC12 cells were cocultured with unlabeled UV‐treated or untreated PC12 cells for 24 h. Then DiD‐RAW264.7 cells were added and cultured for 24 h in the presence or absence of 50 nM cytoD. The phagocytosis of CTG‐PC12 cells was measured by flow cytometry and expressed as relative phagocytosis after normalization with control (n = 4).
    Figure Legend Snippet: Inhibition of TNT formation deceased phagocytosis of viable PC12 cells. (A) Dose‐dependent inhibition of TNT number in PC12 cells after 24 h treatment with cytoD. Scale bars, 20 μm. (B) PS‐positive TNTs in PC12 cells were abolished by 24 h treatment of 50 nM cytoD. Scale bars, 30 μm. (C) 50 nM cytoD did not inhibit phagocytosis of apoptotic PC12 cells. DiD‐RAW264.7 cells were cocultured with CTG‐stained untreated or UV‐treated PC12 cells in the absence and presence of 50 nM cytoD for 24 h, resp. Phagocytosis was measured by flow cytometry, and expressed as the percentage of CTG/DiD double‐positive RAW264.7 cells as a subpopulation of all DiD‐positive RAW264.7 cells. (D) Phagocytosis of viable PC12 cells was inhibited by 50 nM cytoD. CTG‐PC12 cells were cocultured with unlabeled UV‐treated or untreated PC12 cells for 24 h. Then DiD‐RAW264.7 cells were added and cultured for 24 h in the presence or absence of 50 nM cytoD. The phagocytosis of CTG‐PC12 cells was measured by flow cytometry and expressed as relative phagocytosis after normalization with control (n = 4).

    Techniques Used: Inhibition, CTG Assay, Staining, Flow Cytometry, Cytometry, Cell Culture

    6) Product Images from "Rearrangement of microtubule network under biochemical and mechanical stimulations"

    Article Title: Rearrangement of microtubule network under biochemical and mechanical stimulations

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2013.02.014

    Elastic modulus of CHO cells treated with nocodazole, trypsin or cytochalasin D. p-values were derived from Microsoft Excel’s Student’s T-test. N=10.
    Figure Legend Snippet: Elastic modulus of CHO cells treated with nocodazole, trypsin or cytochalasin D. p-values were derived from Microsoft Excel’s Student’s T-test. N=10.

    Techniques Used: Derivative Assay

    7) Product Images from "Processing of Mycobacterium tuberculosis Bacilli by Human Monocytes for CD4+ ?? and ?? T Cells: Role of Particulate Antigen"

    Article Title: Processing of Mycobacterium tuberculosis Bacilli by Human Monocytes for CD4+ ?? and ?? T Cells: Role of Particulate Antigen

    Journal: Infection and Immunity

    doi:

    Requirement for phagocytosis by monocytes of M. tuberculosis bacilli for activation of CD4 + and γδ T cells. Monocytes (MO) were incubated with M. tuberculosis (MTB) in the presence (CCD-MO+MTB) or absence (MO+MTB) of cytochalasin D (CCD) for 120 min, after which they were washed and irradiated before coculture with CD4 + (A) and γδ (B) T-cell lines in a proliferation assay. Results are means and standard deviations of triplicate wells and are representative of five experiments. Monocyte targets were from the same donor as the CD4 + T-cell line, and the γδ T-cell line was derived from an unrelated donor.
    Figure Legend Snippet: Requirement for phagocytosis by monocytes of M. tuberculosis bacilli for activation of CD4 + and γδ T cells. Monocytes (MO) were incubated with M. tuberculosis (MTB) in the presence (CCD-MO+MTB) or absence (MO+MTB) of cytochalasin D (CCD) for 120 min, after which they were washed and irradiated before coculture with CD4 + (A) and γδ (B) T-cell lines in a proliferation assay. Results are means and standard deviations of triplicate wells and are representative of five experiments. Monocyte targets were from the same donor as the CD4 + T-cell line, and the γδ T-cell line was derived from an unrelated donor.

    Techniques Used: Activation Assay, Incubation, Irradiation, Proliferation Assay, Derivative Assay

    8) Product Images from "Adipose stem cell-derived nanovesicles inhibit emphysema primarily via an FGF2-dependent pathway"

    Article Title: Adipose stem cell-derived nanovesicles inhibit emphysema primarily via an FGF2-dependent pathway

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2016.127

    Internalization mechanisms of ASC-derived artificial nanovesicles. ( a ) Confocal microscopy of MLE12 cells incubated with/without a 10 μg ml −1 dose of ASC-derived artificial nanovesicles from ASCs for 2 h on a 35 mm tissue culture dish. ((−) Ctrl: without artificial nanovesicles, (+) Ctrl: with nanovesicles, LY294002 (1 μ M ), cytochalasin D (0.2 μ M ), chloropromazine (1 μg ml −1 ), or heparin (10 μg ml −1 ): cells were treated with each drug for 30 min before artificial nanovesicle treatment). ( b ) Confocal microscopy of MLE12 cells incubated with a 10 μg ml −1 dose of ASC-derived artificial nanovesicles from ASCs for 2 h on a 27 mm collagen coated tissue culture dish ((+) Ctrl: with nanovesicles, Heparin: heparin (10 μg ml −1 ) treatment for 30 min before artificial nanovesicle treatment).
    Figure Legend Snippet: Internalization mechanisms of ASC-derived artificial nanovesicles. ( a ) Confocal microscopy of MLE12 cells incubated with/without a 10 μg ml −1 dose of ASC-derived artificial nanovesicles from ASCs for 2 h on a 35 mm tissue culture dish. ((−) Ctrl: without artificial nanovesicles, (+) Ctrl: with nanovesicles, LY294002 (1 μ M ), cytochalasin D (0.2 μ M ), chloropromazine (1 μg ml −1 ), or heparin (10 μg ml −1 ): cells were treated with each drug for 30 min before artificial nanovesicle treatment). ( b ) Confocal microscopy of MLE12 cells incubated with a 10 μg ml −1 dose of ASC-derived artificial nanovesicles from ASCs for 2 h on a 27 mm collagen coated tissue culture dish ((+) Ctrl: with nanovesicles, Heparin: heparin (10 μg ml −1 ) treatment for 30 min before artificial nanovesicle treatment).

    Techniques Used: Derivative Assay, Confocal Microscopy, Incubation

    9) Product Images from "Excess α-synuclein compromises phagocytosis in iPSC-derived macrophages"

    Article Title: Excess α-synuclein compromises phagocytosis in iPSC-derived macrophages

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09362-3

    High levels of αS reduce phagocytosis in pMac. Phagocytosis was measured by uptake of fluorescent zymosan by pMac. ( A ) Z-projection of confocal images (scale bar = 20 µm). ( B ) representative FACS plots of zymosan uptake (black line, untreated with cytochalasin D; gray, cytochalasin D-treated). ( C ) Quantification of ( B ) (3 independent experiments). ( D ) Incubation with monomeric αS for different lengths of time, then challenge with zymosan (one line). ( E ) Incubation with different concentrations of monomeric αS (24 hr), then challenge with zymosan. Values normalized to untreated mean for each of 3 independent experiments ( C , E ). Statistical analyses, one way ANOVA with Dunnett’s multiple comparisons test. Also see Figure   S4A–C .
    Figure Legend Snippet: High levels of αS reduce phagocytosis in pMac. Phagocytosis was measured by uptake of fluorescent zymosan by pMac. ( A ) Z-projection of confocal images (scale bar = 20 µm). ( B ) representative FACS plots of zymosan uptake (black line, untreated with cytochalasin D; gray, cytochalasin D-treated). ( C ) Quantification of ( B ) (3 independent experiments). ( D ) Incubation with monomeric αS for different lengths of time, then challenge with zymosan (one line). ( E ) Incubation with different concentrations of monomeric αS (24 hr), then challenge with zymosan. Values normalized to untreated mean for each of 3 independent experiments ( C , E ). Statistical analyses, one way ANOVA with Dunnett’s multiple comparisons test. Also see Figure  S4A–C .

    Techniques Used: FACS, Incubation

    10) Product Images from "Targeting of a Tropomyosin Isoform to Short Microfilaments Associated with the Golgi Complex"

    Article Title: Targeting of a Tropomyosin Isoform to Short Microfilaments Associated with the Golgi Complex

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-03-0176

    Tm5NM-2 localization is sensitive to nocodazole but not cytochalasin D treatment. Exposure of NIH3T3 cells to 30 μM nocodazole for 1 h resulted in dispersal of Tm5NM-2 throughout the cytoplasm into small puncta (B), whereas the vehicle had no effect (A). In contrast, treatment with 2 μM cytochalasin D for 1 h had no impact on the organization of Tm5NM-2 (D) compared with treatment with vehicle alone (C). Scale bars, 15 μm.
    Figure Legend Snippet: Tm5NM-2 localization is sensitive to nocodazole but not cytochalasin D treatment. Exposure of NIH3T3 cells to 30 μM nocodazole for 1 h resulted in dispersal of Tm5NM-2 throughout the cytoplasm into small puncta (B), whereas the vehicle had no effect (A). In contrast, treatment with 2 μM cytochalasin D for 1 h had no impact on the organization of Tm5NM-2 (D) compared with treatment with vehicle alone (C). Scale bars, 15 μm.

    Techniques Used:

    11) Product Images from "Interferon-Gamma and Nitric Oxide Synthase 2 Mediate the Aggregation of Resident Adherent Peritoneal Exudate Cells: Implications for the Host Response to Pathogens"

    Article Title: Interferon-Gamma and Nitric Oxide Synthase 2 Mediate the Aggregation of Resident Adherent Peritoneal Exudate Cells: Implications for the Host Response to Pathogens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0128301

    Actin and Tubulin stabilization contribute to Ifnγ induced aggregation of APECs. Fluorescence microscopic images acquired at a magnification of 63X with the scale bar representing 10 μm of F-Actin (green) and α-Tubulin (red) in C57BL/6 APECs treated with 10 μM Cyt D and 1 μg/ml of Col respectively for 6 h (A). Yellow and white arrows indicate cortical arrangement of F-Actin and α-Tubulin respectively. Thin white arrows indicate the cell boundary while the fatter white arrows are used to highlight the shrinkage of the α-Tubulin network upon Col treatment when compared to the untreated control C57BL/6 APECs. The amount of nitrite in the supernatant (B) produced by APECs from C57BL/6 mice upon treatment with 25 U/ml of Ifnγ in the absence or presence indicated doses of inhibitors Cyt D (μM) and Col (μg/ml). The number of cell aggregates (C) of APECs from C57BL/6 mice upon treatment with 25 U/ml of Ifnγ in the absence or presence of indicated doses of inhibitors Cyt D (μM) and Col (μg/ml). The amounts of E-Selectin (D) and CD11b (E) on the cell surface of C57BL/6 APECs treated with 25 U/ml of Ifnγ for 36 h, post 6 h of pretreatment without or with Cyt D (10 μM) and Col (1 μg/ml). The data is represented as mean ± S.E from three independent experiments. The velocity of APECs from C57BL/6 and  Nos2 -/-  mice treated without or with 25 U/ml of Ifnγ between 18–24 h of addition (F). The velocity of APECs from C57BL/6 pretreated for 6 h with Cyt D (10 μM) and Col (1 μg/ml) before tracking them from 6–12 h post treatment. Also, the velocity of  Nos2 -/-  APECs pretreated in the presence of 5 μM of SNAP (SNAP lo ) and 100 μM of SNAP (SNAP hi ) for 12 h and without or with 25 U/ml of Ifnγ before tracking cells 18–24 h post Ifnγ addition. The velocity for each of the above mentioned conditions are represented as percentage velocity with respect to average velocity exhibited by untreated C57BL/6 APECs. The data is representative of two independent experiments. Significance is represented as * when compared to untreated controls and # when compared to Ifnγ treated C57BL/6 APECs respectively. The significance with respect to untreated controls, Ifnγ treated C57BL/6 APECs controls. untreated  Nos2 -/-  and SNAP lo  alone  Nos2 -/-  APECs controls are represented as *, θ, Δ, and # respectively.
    Figure Legend Snippet: Actin and Tubulin stabilization contribute to Ifnγ induced aggregation of APECs. Fluorescence microscopic images acquired at a magnification of 63X with the scale bar representing 10 μm of F-Actin (green) and α-Tubulin (red) in C57BL/6 APECs treated with 10 μM Cyt D and 1 μg/ml of Col respectively for 6 h (A). Yellow and white arrows indicate cortical arrangement of F-Actin and α-Tubulin respectively. Thin white arrows indicate the cell boundary while the fatter white arrows are used to highlight the shrinkage of the α-Tubulin network upon Col treatment when compared to the untreated control C57BL/6 APECs. The amount of nitrite in the supernatant (B) produced by APECs from C57BL/6 mice upon treatment with 25 U/ml of Ifnγ in the absence or presence indicated doses of inhibitors Cyt D (μM) and Col (μg/ml). The number of cell aggregates (C) of APECs from C57BL/6 mice upon treatment with 25 U/ml of Ifnγ in the absence or presence of indicated doses of inhibitors Cyt D (μM) and Col (μg/ml). The amounts of E-Selectin (D) and CD11b (E) on the cell surface of C57BL/6 APECs treated with 25 U/ml of Ifnγ for 36 h, post 6 h of pretreatment without or with Cyt D (10 μM) and Col (1 μg/ml). The data is represented as mean ± S.E from three independent experiments. The velocity of APECs from C57BL/6 and Nos2 -/- mice treated without or with 25 U/ml of Ifnγ between 18–24 h of addition (F). The velocity of APECs from C57BL/6 pretreated for 6 h with Cyt D (10 μM) and Col (1 μg/ml) before tracking them from 6–12 h post treatment. Also, the velocity of Nos2 -/- APECs pretreated in the presence of 5 μM of SNAP (SNAP lo ) and 100 μM of SNAP (SNAP hi ) for 12 h and without or with 25 U/ml of Ifnγ before tracking cells 18–24 h post Ifnγ addition. The velocity for each of the above mentioned conditions are represented as percentage velocity with respect to average velocity exhibited by untreated C57BL/6 APECs. The data is representative of two independent experiments. Significance is represented as * when compared to untreated controls and # when compared to Ifnγ treated C57BL/6 APECs respectively. The significance with respect to untreated controls, Ifnγ treated C57BL/6 APECs controls. untreated Nos2 -/- and SNAP lo alone Nos2 -/- APECs controls are represented as *, θ, Δ, and # respectively.

    Techniques Used: Fluorescence, Produced, Mouse Assay

    12) Product Images from "Human Mast Cell Activation by Staphylococcus aureus: Interleukin-8 and Tumor Necrosis Factor Alpha Release and the Role of Toll-Like Receptor 2 and CD48 Molecules ▿"

    Article Title: Human Mast Cell Activation by Staphylococcus aureus: Interleukin-8 and Tumor Necrosis Factor Alpha Release and the Role of Toll-Like Receptor 2 and CD48 Molecules ▿

    Journal:

    doi: 10.1128/IAI.00270-08

    Cytochalasin D inhibits S. aureus invasion of CBMC. CBMC treated with cytochalasin D for 60 min before infection were incubated with S. aureus strains (MOI of 10) for 180 min at 37°C, and the percentage of internalized bacteria was evaluated.
    Figure Legend Snippet: Cytochalasin D inhibits S. aureus invasion of CBMC. CBMC treated with cytochalasin D for 60 min before infection were incubated with S. aureus strains (MOI of 10) for 180 min at 37°C, and the percentage of internalized bacteria was evaluated.

    Techniques Used: Infection, Incubation

    IL-8 (A) and TNF-α (B) release by CBMC is dependent on  S. aureus  internalization. CBMC were left uninfected, infected with  S. aureus  at 37°C for 180 min in the presence or absence of cytochalasin D, or incubated with PGN (10 μg
    Figure Legend Snippet: IL-8 (A) and TNF-α (B) release by CBMC is dependent on S. aureus internalization. CBMC were left uninfected, infected with S. aureus at 37°C for 180 min in the presence or absence of cytochalasin D, or incubated with PGN (10 μg

    Techniques Used: Infection, Incubation

    13) Product Images from "Unbiased High-Precision Cell Mechanical Measurements with Microconstrictions"

    Article Title: Unbiased High-Precision Cell Mechanical Measurements with Microconstrictions

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2017.02.018

    ( a – d ) Measurement of a mixed K562 leukemia cell population, containing 50% untreated cells and 50% cells treated with cytoD (10 μ M, 30 min) and stained with calcein (250 nM, 30 min). ( a ) Histogram of average fluorescence intensities per cell pixel. ( Dashed green line ) Fluorescence threshold for separating cells into a zero and a high fluorescence intensity group. ( b ) Exemplary histogram-matched scatter plots of t entry versus ε max /Δ p for cells with zero fluorescence (nontreated) and high fluorescence (cytoD and calcein-treated) after histogram matching. ( Black lines ) Fit of Eq. 1 to the data. ( c ) Cell elastic modulus and ( d ) cell fluidity for the two groups. ( e – h ) Measurement of lamin A-GFP-transfected K562 leukemia cells. ( e ) Histogram of average fluorescence intensities per cell pixel. ( Dashed green lines ) Fluorescence threshold for separating cells into low, medium, and high fluorescence intensity groups. ( f ) Exemplary histogram-matched scatter plots of t entry versus ε max /Δ p for cells with low, medium, and high expression levels of lamin A-GFP. ( Black lines ) Fit of Eq. 1 to the data. ( g ) Cell elastic moduli and ( h ) cell fluidity for the three groups. Significant ( p
    Figure Legend Snippet: ( a – d ) Measurement of a mixed K562 leukemia cell population, containing 50% untreated cells and 50% cells treated with cytoD (10 μ M, 30 min) and stained with calcein (250 nM, 30 min). ( a ) Histogram of average fluorescence intensities per cell pixel. ( Dashed green line ) Fluorescence threshold for separating cells into a zero and a high fluorescence intensity group. ( b ) Exemplary histogram-matched scatter plots of t entry versus ε max /Δ p for cells with zero fluorescence (nontreated) and high fluorescence (cytoD and calcein-treated) after histogram matching. ( Black lines ) Fit of Eq. 1 to the data. ( c ) Cell elastic modulus and ( d ) cell fluidity for the two groups. ( e – h ) Measurement of lamin A-GFP-transfected K562 leukemia cells. ( e ) Histogram of average fluorescence intensities per cell pixel. ( Dashed green lines ) Fluorescence threshold for separating cells into low, medium, and high fluorescence intensity groups. ( f ) Exemplary histogram-matched scatter plots of t entry versus ε max /Δ p for cells with low, medium, and high expression levels of lamin A-GFP. ( Black lines ) Fit of Eq. 1 to the data. ( g ) Cell elastic moduli and ( h ) cell fluidity for the three groups. Significant ( p

    Techniques Used: Staining, Fluorescence, Transfection, Expressing

    14) Product Images from "Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion"

    Article Title: Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion

    Journal: Nature

    doi: 10.1038/nature13242

    Amoebic trogocytosis occurs with red blood cells, contributes to intestinal invasion, and promotes enhanced cell killing a, DiD-labeled red blood cells (RBC) and CMFDA-labeled amoebae (A); ingested bites (arrows) and a fragment remains extracellular (0:51). Bar, 5 μm. Images are representative of three independent experiments. b, Example 3-D reconstruction with DiD-labeled red blood cells and CMFDA-labeled amoebae; biting (arrows) and a fragment remains extracellular (3:25). Images are representative of three independent experiments. c, Planes from 4-D two-photon microscopy with mouse intestinal tissue (yellow); calcein violet-labeled amoebae (blue) are ingesting bites (arrowheads). Images are representative of three independent experiments. d, Amoebae treated with wortmannin or cytochalasin D were incubated with tissue. Invasion depth, means and standard deviations from biological replicates (at least 40 cells/treatment); P-values are indicated. e–g, Amoebae were incubated with or without human Jurkat cells (“primed”), prior to incubation with labeled human Jurkat cells. e, Measurement of internalization. f, Measurement of fragmentation. g, Detection of dead cells. Means and standard deviations for biological duplicates (10,000 events/each). P-values from t -tests: *
    Figure Legend Snippet: Amoebic trogocytosis occurs with red blood cells, contributes to intestinal invasion, and promotes enhanced cell killing a, DiD-labeled red blood cells (RBC) and CMFDA-labeled amoebae (A); ingested bites (arrows) and a fragment remains extracellular (0:51). Bar, 5 μm. Images are representative of three independent experiments. b, Example 3-D reconstruction with DiD-labeled red blood cells and CMFDA-labeled amoebae; biting (arrows) and a fragment remains extracellular (3:25). Images are representative of three independent experiments. c, Planes from 4-D two-photon microscopy with mouse intestinal tissue (yellow); calcein violet-labeled amoebae (blue) are ingesting bites (arrowheads). Images are representative of three independent experiments. d, Amoebae treated with wortmannin or cytochalasin D were incubated with tissue. Invasion depth, means and standard deviations from biological replicates (at least 40 cells/treatment); P-values are indicated. e–g, Amoebae were incubated with or without human Jurkat cells (“primed”), prior to incubation with labeled human Jurkat cells. e, Measurement of internalization. f, Measurement of fragmentation. g, Detection of dead cells. Means and standard deviations for biological duplicates (10,000 events/each). P-values from t -tests: *

    Techniques Used: Labeling, Microscopy, Incubation

    15) Product Images from "Periodic actin structures in neuronal axons are required to maintain microtubules"

    Article Title: Periodic actin structures in neuronal axons are required to maintain microtubules

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E16-10-0727

    Combined loss of F-actin and Shot reduces axon numbers. (A–D’) Primary neurons at 8 HIV from wild-type (wt) or shot mutant embryos at 8 HIV treated for 4 h with either DMSO or CytoD as indicated on the left and stained for tubulin (green, white on right) and horseradish peroxidase (magenta). CytoD treatment alone causes gaps in the axonal tubulin staining (B; arrowheads, MT gaps); Shot deficiency causes MT disorganization (C; double arrow); and combination of shot with CytoD has a detrimental effect on axons (D). (E) Statistical analysis of neurons with/without axons; numbers in bars refer to analyzed neurons; all data were compared with DMSO controls via chi-squared analysis (n.s., p > 0.050; * p
    Figure Legend Snippet: Combined loss of F-actin and Shot reduces axon numbers. (A–D’) Primary neurons at 8 HIV from wild-type (wt) or shot mutant embryos at 8 HIV treated for 4 h with either DMSO or CytoD as indicated on the left and stained for tubulin (green, white on right) and horseradish peroxidase (magenta). CytoD treatment alone causes gaps in the axonal tubulin staining (B; arrowheads, MT gaps); Shot deficiency causes MT disorganization (C; double arrow); and combination of shot with CytoD has a detrimental effect on axons (D). (E) Statistical analysis of neurons with/without axons; numbers in bars refer to analyzed neurons; all data were compared with DMSO controls via chi-squared analysis (n.s., p > 0.050; * p

    Techniques Used: Mutagenesis, Staining

    Effects of actin manipulations on filopodial length. (A–F’) Filopodial length phenotypes in DMSO-treated wild-type primary neurons or neurons treated with drugs or being mutant, as indicated; cells are double-labeled for actin (green in top row, white in bottom row) and tubulin (magenta in top row); drug treatments: 800 nM CytD for 4 h, 200 nM LatA for 1 h. (G) Quantifications of filopodia length caused by drug treatment or mutations shown at the top (all normalized and compared with DMSO-treated controls); numbers above the bars indicate the number of filopodia analyzed in each experiment; note that filopodia were completely absent in all cases of CytoD and LatA treatment. p values were calculated using the Mann–Whitney rank sum test (n.s., p > 0.05; *** p
    Figure Legend Snippet: Effects of actin manipulations on filopodial length. (A–F’) Filopodial length phenotypes in DMSO-treated wild-type primary neurons or neurons treated with drugs or being mutant, as indicated; cells are double-labeled for actin (green in top row, white in bottom row) and tubulin (magenta in top row); drug treatments: 800 nM CytD for 4 h, 200 nM LatA for 1 h. (G) Quantifications of filopodia length caused by drug treatment or mutations shown at the top (all normalized and compared with DMSO-treated controls); numbers above the bars indicate the number of filopodia analyzed in each experiment; note that filopodia were completely absent in all cases of CytoD and LatA treatment. p values were calculated using the Mann–Whitney rank sum test (n.s., p > 0.05; *** p

    Techniques Used: Mutagenesis, Labeling, MANN-WHITNEY

    F-actin has MT stabilizing roles. (A–C) Primary wild-type neurons at 8 HIV stained with phalloidin for actin (magenta) and against tubulin (green), either untreated (wt) or treated with LatA or CytoD (asterisks, cell bodies; arrows, axon tips; arrowheads, MT gaps); images show gap phenotypes in C. (D) Quantification plotting axons with gaps normalized to 4-h DMSO-treated controls; note that washout of CytoD can revert the phenotype (red arrow; bar on its right shows that CytoD still has gap-inducing activity during the washout period) and that cotreatment with nocodazole (Noco) enhances the phenotype (highest bar); numbers in bars refer to analyzed neurons; all data were compared with DMSO controls via chi-squared analysis (n.s., p > 0.05, *** p
    Figure Legend Snippet: F-actin has MT stabilizing roles. (A–C) Primary wild-type neurons at 8 HIV stained with phalloidin for actin (magenta) and against tubulin (green), either untreated (wt) or treated with LatA or CytoD (asterisks, cell bodies; arrows, axon tips; arrowheads, MT gaps); images show gap phenotypes in C. (D) Quantification plotting axons with gaps normalized to 4-h DMSO-treated controls; note that washout of CytoD can revert the phenotype (red arrow; bar on its right shows that CytoD still has gap-inducing activity during the washout period) and that cotreatment with nocodazole (Noco) enhances the phenotype (highest bar); numbers in bars refer to analyzed neurons; all data were compared with DMSO controls via chi-squared analysis (n.s., p > 0.05, *** p

    Techniques Used: Staining, Activity Assay

    Live recordings of wild-type and shot -deficient primary neurons expressing EB1::GFP. (A, B) Stills of movies of neurons at 6 HIV taken at three time points (0, 30, and 60 min after treatment), where each still is a projection of four images 3 s apart and alternately colored green and magenta to indicate the movement of EB1::GFP comets; note that comets come to a halt and vanish only in shot -mutant neurons. Measurements of comet numbers (C, D, F, G) and velocities (E, H) of EB1::GFP comets in wild-type and shot 3 mutant neurons upon treatment with 0.8 µM CytoD, 1.6 µM CytoD, or 200 nM LatA, as indicated in box at the bottom; velocity of wt is 0.154 µm/s ± 0.01 SEM; p values in C–H were calculated as explained in Materials and Methods ; two-color code in p values of C and E indicates which curves were compared; for detailed data, see Supplemental Figure S1 and Table S1, p. 5. Scale bar, 5 μm.
    Figure Legend Snippet: Live recordings of wild-type and shot -deficient primary neurons expressing EB1::GFP. (A, B) Stills of movies of neurons at 6 HIV taken at three time points (0, 30, and 60 min after treatment), where each still is a projection of four images 3 s apart and alternately colored green and magenta to indicate the movement of EB1::GFP comets; note that comets come to a halt and vanish only in shot -mutant neurons. Measurements of comet numbers (C, D, F, G) and velocities (E, H) of EB1::GFP comets in wild-type and shot 3 mutant neurons upon treatment with 0.8 µM CytoD, 1.6 µM CytoD, or 200 nM LatA, as indicated in box at the bottom; velocity of wt is 0.154 µm/s ± 0.01 SEM; p values in C–H were calculated as explained in Materials and Methods ; two-color code in p values of C and E indicates which curves were compared; for detailed data, see Supplemental Figure S1 and Table S1, p. 5. Scale bar, 5 μm.

    Techniques Used: Expressing, Mutagenesis

    16) Product Images from "p62 and NDP52 Proteins Target Intracytosolic Shigella and Listeria"

    Article Title: p62 and NDP52 Proteins Target Intracytosolic Shigella and Listeria

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.223610

    NDP52 and p62 recruitment to Shigella depends on actin and Shigella IcsB. A , HeLa cells were infected with S. flexneri , treated with dimethyl sulfoxide ( DMSO ) or cytochalasin D ( CytD ), and after 4 h 40 min were fixed and labeled for quantitative microscopy.
    Figure Legend Snippet: NDP52 and p62 recruitment to Shigella depends on actin and Shigella IcsB. A , HeLa cells were infected with S. flexneri , treated with dimethyl sulfoxide ( DMSO ) or cytochalasin D ( CytD ), and after 4 h 40 min were fixed and labeled for quantitative microscopy.

    Techniques Used: Infection, Labeling, Microscopy

    17) Product Images from "The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii"

    Article Title: The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002007

    Parasites lacking RON8 are deficient in invasion likely through increased detachment from host cells. A ) Quantification of invasion using red/green assays demonstrates a substantial invasion defect for Δron8 parasites that is rescued upon complementation. Green bars represent internal/penetrated parasites, while red bars depict attached/extracellular parasites for wildtype, Δron8 , or R8c strains allowed to invade fibroblast monolayers for 1 hour. For each strain, at least 250 total parasites were counted from nine random fields per sample, and values are presented as internal (Int) or external (Ext) parasites per field. Data are mean values +/− SEM (error bars) for two independent experiments performed in triplicate. The asterisk indicates that parasite penetration is significantly lower (p value = 0.0245 using a Student's two-tailed t-test) in Δ ron8 parasites compared to wildtype. B ) Initial stages of attachment are unaffected in Δ ron8 parasites. Equal numbers of wildtype, Δron8 , or R8c parasites were preincubated with 1 µM cytochalasin D for 15 min prior to incubation with host fibroblasts in the presence of cytochalasin D, then fixed and stained in detergent-free conditions with rabbit antisera against SAG1. For each strain, values are displayed as total numbers of parasites counted divided over nine random fields. The data is expressed as mean values +/− SEM for two independent experiments performed in triplicate.
    Figure Legend Snippet: Parasites lacking RON8 are deficient in invasion likely through increased detachment from host cells. A ) Quantification of invasion using red/green assays demonstrates a substantial invasion defect for Δron8 parasites that is rescued upon complementation. Green bars represent internal/penetrated parasites, while red bars depict attached/extracellular parasites for wildtype, Δron8 , or R8c strains allowed to invade fibroblast monolayers for 1 hour. For each strain, at least 250 total parasites were counted from nine random fields per sample, and values are presented as internal (Int) or external (Ext) parasites per field. Data are mean values +/− SEM (error bars) for two independent experiments performed in triplicate. The asterisk indicates that parasite penetration is significantly lower (p value = 0.0245 using a Student's two-tailed t-test) in Δ ron8 parasites compared to wildtype. B ) Initial stages of attachment are unaffected in Δ ron8 parasites. Equal numbers of wildtype, Δron8 , or R8c parasites were preincubated with 1 µM cytochalasin D for 15 min prior to incubation with host fibroblasts in the presence of cytochalasin D, then fixed and stained in detergent-free conditions with rabbit antisera against SAG1. For each strain, values are displayed as total numbers of parasites counted divided over nine random fields. The data is expressed as mean values +/− SEM for two independent experiments performed in triplicate.

    Techniques Used: Significance Assay, Two Tailed Test, Incubation, Staining

    18) Product Images from "Requirement for an Intact T-Cell Actin and Tubulin Cytoskeleton for Efficient Assembly and Spread of Human Immunodeficiency Virus Type 1 ▿"

    Article Title: Requirement for an Intact T-Cell Actin and Tubulin Cytoskeleton for Efficient Assembly and Spread of Human Immunodeficiency Virus Type 1 ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01469-06

    Cytoskeletal inhibitors reduce cell-cell spread, as measured by qPCR. Equal numbers of Jurkat LAI  cells and uninfected target cells were mixed and incubated for 1, 3, and 6 h prior to cell lysis and purification of total cellular DNA. qPCR using pol primers was performed to measure de novo viral DNA synthesis. Data were normalized to HSA and are shown as ratios of HIV-1 DNA to HSA DNA. The data obtained with A2.01 target cells were used as baseline HIV-1 DNA synthesis at the different time points and were subtracted from the signal with A3.01 target cells. (A) Jurkat LAI  cells were either left untreated or incubated with Q4120 or AMD3100 prior to being mixed with A3.01 target cells to inhibit Env-CD4 or Env-CXCR4 interactions, respectively. (B) Jurkat LAI  cells were either left untreated or incuba ted with an inhibitor of actin remodeling (latrunculin, cytochalasin D, or jasplakinolide) prior to being washed and mixed with A3.01 cells. (C) Jurkat LAI  cells were either left untreated or incubated with an inhibitor of microtubule remodeling (colchicine or nocodazole). Bars represent the means for triplicate samples plus 1 SD.
    Figure Legend Snippet: Cytoskeletal inhibitors reduce cell-cell spread, as measured by qPCR. Equal numbers of Jurkat LAI cells and uninfected target cells were mixed and incubated for 1, 3, and 6 h prior to cell lysis and purification of total cellular DNA. qPCR using pol primers was performed to measure de novo viral DNA synthesis. Data were normalized to HSA and are shown as ratios of HIV-1 DNA to HSA DNA. The data obtained with A2.01 target cells were used as baseline HIV-1 DNA synthesis at the different time points and were subtracted from the signal with A3.01 target cells. (A) Jurkat LAI cells were either left untreated or incubated with Q4120 or AMD3100 prior to being mixed with A3.01 target cells to inhibit Env-CD4 or Env-CXCR4 interactions, respectively. (B) Jurkat LAI cells were either left untreated or incuba ted with an inhibitor of actin remodeling (latrunculin, cytochalasin D, or jasplakinolide) prior to being washed and mixed with A3.01 cells. (C) Jurkat LAI cells were either left untreated or incubated with an inhibitor of microtubule remodeling (colchicine or nocodazole). Bars represent the means for triplicate samples plus 1 SD.

    Techniques Used: Real-time Polymerase Chain Reaction, Incubation, Lysis, Purification, DNA Synthesis

    Effects of cytoskeletal inhibitors on HIV-1 release and viral infectivity. Jurkat LAI  cells were either left untreated or treated with 1 μM of nocodazole, colchicine, cytochalasin D, or latrunculin for 3 h at 37°C. (A) Cell and viral lysates from untreated or inhibitor-treated cells were separated by SDS-PAGE, and HIV-1 Gag was detected by Western blotting. C, lysates prepared from cell pellets; V, lysates prepared from cell-free virus-containing supernatants. For each condition, 20 μg of cell lysate was used and an equal volume of viral lysate was loaded to allow a direct comparison of virus outputs. The Gag polyprotein p55 and the capsid protein p24 are indicated. (B) Densitometer analysis was performed on Gag p55 and p24 bands from nonsaturated Western blots, and for each treatment condition, the ratio of total cell-free to total cell-associated Gag was calculated. Values are the averages for three independent virus release assays, and the error bars show the SEM. (C) Env incorporation into HIV-1 virions from cells treated with cytoskeletal inhibitors. Supernatants were harvested from Jurkat LAI  cells that were either left untreated or treated with cytoskeletal inhibitors for 3 h, and virions were concentrated by centrifugation. Viral lysates were separated by SDS-PAGE, and HIV-1 Env was detected by Western blotting with rabbit anti-gp120 serum. A representative blot is shown. (D) Densitometer analysis was performed on gp120 bands from nonsaturated blots. Values are the average pixel intensities of individual bands corresponding to gp120 and are the averages for two independent virus release experiments. Error bars show the SEM. (E) Infectivity assay on viral supernatants harvested from inhibitor-treated cells. Jurkat LAI  cells were either left untreated (white bar) or treated for 3 h with cytoskeletal inhibitors (gray bars). The cells were then washed to remove inhibitors and incubated for 1 h at 37°C, and virus-containing supernatants were collected and used to infect HeLa reporter cells. Viral infectivity was measured by performing a β-galactosidase assay, and values show the percentages of infectivity relative to that of the untreated control, which was normalized to 100%. Data are the averages for two independent virus release assays, and error bars show the SEM. (F) After 3 h of incubation in the presence or absence of inhibitors, the number of viable Jurkat LAI  cells was calculated by trypan blue exclusion. Error bars show the standard deviations (SD) for a single experiment performed in triplicate. (G) The metabolic activity of treated or untreated cells was determined using a nonradioactive colorimetric cellular proliferation assay (Promega) that measures bioreduction of the MTS tetrazolium compound. The absorbance at 490 nm was measured, and values are shown for each inhibitor and the untreated control. Error bars show the SD.
    Figure Legend Snippet: Effects of cytoskeletal inhibitors on HIV-1 release and viral infectivity. Jurkat LAI cells were either left untreated or treated with 1 μM of nocodazole, colchicine, cytochalasin D, or latrunculin for 3 h at 37°C. (A) Cell and viral lysates from untreated or inhibitor-treated cells were separated by SDS-PAGE, and HIV-1 Gag was detected by Western blotting. C, lysates prepared from cell pellets; V, lysates prepared from cell-free virus-containing supernatants. For each condition, 20 μg of cell lysate was used and an equal volume of viral lysate was loaded to allow a direct comparison of virus outputs. The Gag polyprotein p55 and the capsid protein p24 are indicated. (B) Densitometer analysis was performed on Gag p55 and p24 bands from nonsaturated Western blots, and for each treatment condition, the ratio of total cell-free to total cell-associated Gag was calculated. Values are the averages for three independent virus release assays, and the error bars show the SEM. (C) Env incorporation into HIV-1 virions from cells treated with cytoskeletal inhibitors. Supernatants were harvested from Jurkat LAI cells that were either left untreated or treated with cytoskeletal inhibitors for 3 h, and virions were concentrated by centrifugation. Viral lysates were separated by SDS-PAGE, and HIV-1 Env was detected by Western blotting with rabbit anti-gp120 serum. A representative blot is shown. (D) Densitometer analysis was performed on gp120 bands from nonsaturated blots. Values are the average pixel intensities of individual bands corresponding to gp120 and are the averages for two independent virus release experiments. Error bars show the SEM. (E) Infectivity assay on viral supernatants harvested from inhibitor-treated cells. Jurkat LAI cells were either left untreated (white bar) or treated for 3 h with cytoskeletal inhibitors (gray bars). The cells were then washed to remove inhibitors and incubated for 1 h at 37°C, and virus-containing supernatants were collected and used to infect HeLa reporter cells. Viral infectivity was measured by performing a β-galactosidase assay, and values show the percentages of infectivity relative to that of the untreated control, which was normalized to 100%. Data are the averages for two independent virus release assays, and error bars show the SEM. (F) After 3 h of incubation in the presence or absence of inhibitors, the number of viable Jurkat LAI cells was calculated by trypan blue exclusion. Error bars show the standard deviations (SD) for a single experiment performed in triplicate. (G) The metabolic activity of treated or untreated cells was determined using a nonradioactive colorimetric cellular proliferation assay (Promega) that measures bioreduction of the MTS tetrazolium compound. The absorbance at 490 nm was measured, and values are shown for each inhibitor and the untreated control. Error bars show the SD.

    Techniques Used: Infection, SDS Page, Western Blot, Centrifugation, Incubation, Activity Assay, Proliferation Assay

    19) Product Images from "Listeria monocytogenes Invasion of Epithelial Cells Requires the MEK-1/ERK-2 Mitogen-Activated Protein Kinase Pathway"

    Article Title: Listeria monocytogenes Invasion of Epithelial Cells Requires the MEK-1/ERK-2 Mitogen-Activated Protein Kinase Pathway

    Journal: Infection and Immunity

    doi:

    Various inhibitors reduce L. monocytogenes invasion. HeLa cells were pretreated with 1 μg of cytochalasin D per ml, 100 nM wortmannin, or 250 μM genistein for 30 min prior to bacterial infection. Control cells were treated with 0.2% DMSO in MEM. L. monocytogenes 1/2a 3 was allowed to invade for 60 min before the addition of 100 μg of gentamicin per ml. The monolayers were lysed, and the numbers of intracellular bacteria were determined by plating. The assay was repeated at least three times with similar results, and the graph depicts one representative experiment. Each data point is the mean of data from three wells, and the standard deviation is shown by the error bar.
    Figure Legend Snippet: Various inhibitors reduce L. monocytogenes invasion. HeLa cells were pretreated with 1 μg of cytochalasin D per ml, 100 nM wortmannin, or 250 μM genistein for 30 min prior to bacterial infection. Control cells were treated with 0.2% DMSO in MEM. L. monocytogenes 1/2a 3 was allowed to invade for 60 min before the addition of 100 μg of gentamicin per ml. The monolayers were lysed, and the numbers of intracellular bacteria were determined by plating. The assay was repeated at least three times with similar results, and the graph depicts one representative experiment. Each data point is the mean of data from three wells, and the standard deviation is shown by the error bar.

    Techniques Used: Infection, Standard Deviation

    The effect of various inhibitors on ERK-2 activation. HeLa cells were pretreated with 1 μg of cytochalasin D per ml, 100 nM wortmannin, or 250 μM genistein for 30 min or with 100 μM PD98059 for 60 min prior to bacterial infection. Control cells were treated with 0.2% DMSO in MEM. ERK-2 activity was measured as described in Materials and Methods. The autoradiographs show the level of phosphorylation of MBP by ERK-2. (a) Cytochalasin D does not affect ERK-2 activation by Listeria . (b) Wortmannin does not affect ERK-2 activation by Listeria . (c) Genistein reduces ERK-2 activation by Listeria . (d) PD98059 blocks ERK-2 activation by Listeria . These autoradiographs are each representative of results from three experiments.
    Figure Legend Snippet: The effect of various inhibitors on ERK-2 activation. HeLa cells were pretreated with 1 μg of cytochalasin D per ml, 100 nM wortmannin, or 250 μM genistein for 30 min or with 100 μM PD98059 for 60 min prior to bacterial infection. Control cells were treated with 0.2% DMSO in MEM. ERK-2 activity was measured as described in Materials and Methods. The autoradiographs show the level of phosphorylation of MBP by ERK-2. (a) Cytochalasin D does not affect ERK-2 activation by Listeria . (b) Wortmannin does not affect ERK-2 activation by Listeria . (c) Genistein reduces ERK-2 activation by Listeria . (d) PD98059 blocks ERK-2 activation by Listeria . These autoradiographs are each representative of results from three experiments.

    Techniques Used: Activation Assay, Infection, Activity Assay

    20) Product Images from "MWCNT interactions with protein: surface-induced changes in protein adsorption and the impact of protein corona on cellular uptake and cytotoxicity"

    Article Title: MWCNT interactions with protein: surface-induced changes in protein adsorption and the impact of protein corona on cellular uptake and cytotoxicity

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S191689

    Inhibition of endocytosis by cytochalasin D that attenuates the toxicity of MWCNTs. Notes: Raw264.7 cells preincubated with endocytosis inhibitor, Cytochalasin D (CytoD) for 2 hours, and investigated the cellular uptake, cytotoxicity and pro-inflammatory cytokine secretion after 4 µM CytoD inhibitor pretreatment with bare or BSA coronas MWCNTs. ( A ) Cellular uptake of Raw264.7 cells was analyzed after the treatment of MWCNTs for 6 hours. Cytotoxicity was measured by WST-8 ( B ) and LHD assays ( C ). Levels of IL-1β ( D ) of RAW264.7 cells at 24 hours following the treatment. All assays were performed as described in Materials and methods section. Control cells were not subjected to MWCNTs exposure. * P
    Figure Legend Snippet: Inhibition of endocytosis by cytochalasin D that attenuates the toxicity of MWCNTs. Notes: Raw264.7 cells preincubated with endocytosis inhibitor, Cytochalasin D (CytoD) for 2 hours, and investigated the cellular uptake, cytotoxicity and pro-inflammatory cytokine secretion after 4 µM CytoD inhibitor pretreatment with bare or BSA coronas MWCNTs. ( A ) Cellular uptake of Raw264.7 cells was analyzed after the treatment of MWCNTs for 6 hours. Cytotoxicity was measured by WST-8 ( B ) and LHD assays ( C ). Levels of IL-1β ( D ) of RAW264.7 cells at 24 hours following the treatment. All assays were performed as described in Materials and methods section. Control cells were not subjected to MWCNTs exposure. * P

    Techniques Used: Inhibition

    21) Product Images from "?1 Integrin Expression Increases Susceptibility of Memory B Cells to Epstein-Barr Virus Infection ▿"

    Article Title: ?1 Integrin Expression Increases Susceptibility of Memory B Cells to Epstein-Barr Virus Infection ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02675-09

    Activation of the integrin signal transduction pathway is crucial for EBV entry into memory B cells. (A and B) Inhibition of signaling molecules c-Src, PI3K, and FAK, activated downstream of integrin, impacts the susceptibility of tonsillar memory B cells to EBV infection by B95.8EBfaV-GFP viral supernatants. Inhibitors used were AG-82 (FAK), PP2 (c-Src), wortmannin (PI3K), and Ly-294002 (PI3K). Cytochalasin D was included to show the dependence of infection susceptibility of memory B cells on actin cytoskeleton depolymerization. Cells were counterstained for CD19 and CD27 to identify memory B cells and determine their infection frequency by flow cytometry. Results shown are from one representative experiment of two experiments.
    Figure Legend Snippet: Activation of the integrin signal transduction pathway is crucial for EBV entry into memory B cells. (A and B) Inhibition of signaling molecules c-Src, PI3K, and FAK, activated downstream of integrin, impacts the susceptibility of tonsillar memory B cells to EBV infection by B95.8EBfaV-GFP viral supernatants. Inhibitors used were AG-82 (FAK), PP2 (c-Src), wortmannin (PI3K), and Ly-294002 (PI3K). Cytochalasin D was included to show the dependence of infection susceptibility of memory B cells on actin cytoskeleton depolymerization. Cells were counterstained for CD19 and CD27 to identify memory B cells and determine their infection frequency by flow cytometry. Results shown are from one representative experiment of two experiments.

    Techniques Used: Activation Assay, Transduction, Inhibition, Infection, Flow Cytometry, Cytometry

    22) Product Images from "Human Monoclonal Antibodies Targeting Nonoverlapping Epitopes on Insulin-like Growth Factor II as a Novel Type of Candidate Cancer Therapeutics"

    Article Title: Human Monoclonal Antibodies Targeting Nonoverlapping Epitopes on Insulin-like Growth Factor II as a Novel Type of Candidate Cancer Therapeutics

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-12-0172

    FACS binding of m660–hIGF-II complexes to PMA-stimulated U937 cells. A, expression of FcγRI on U937 cells. The diagram for reference cells that were not stimulated is in purple. The diagram for cells mock-stimulated with PMA solvent dimethyl sulfoxide (DMSO) is in green. The pink, blue, and orange diagrams are for cells stimulated with 10, 20, and 30 ng/mL PMA, respectively. B, binding of biotinylated hIGF-II to U937 cells. The green diagram indicates cells incubated with streptavidin-PE conjugate only. The diagrams for cells incubated with hIGF-II at concentrations of 10, 100, and 1,000 nmol/L are in pink, blue, and orange, respectively. C, interactions of antibody–hIGF-II complexes with U937 cells. The purple diagram is for reference cells incubated with the secondary antibody only. The diagrams for cells incubated with 10 nmol/L antibody alone, 10 nmol/L antibody plus 20 (for monospecific antibodies), or 40 nmol/L (for m660) hIGF-II, and an combination of the antibody, hIGF-II, and 50 μmol/L cytochalasin D are in green, pink, and blue, respectively.
    Figure Legend Snippet: FACS binding of m660–hIGF-II complexes to PMA-stimulated U937 cells. A, expression of FcγRI on U937 cells. The diagram for reference cells that were not stimulated is in purple. The diagram for cells mock-stimulated with PMA solvent dimethyl sulfoxide (DMSO) is in green. The pink, blue, and orange diagrams are for cells stimulated with 10, 20, and 30 ng/mL PMA, respectively. B, binding of biotinylated hIGF-II to U937 cells. The green diagram indicates cells incubated with streptavidin-PE conjugate only. The diagrams for cells incubated with hIGF-II at concentrations of 10, 100, and 1,000 nmol/L are in pink, blue, and orange, respectively. C, interactions of antibody–hIGF-II complexes with U937 cells. The purple diagram is for reference cells incubated with the secondary antibody only. The diagrams for cells incubated with 10 nmol/L antibody alone, 10 nmol/L antibody plus 20 (for monospecific antibodies), or 40 nmol/L (for m660) hIGF-II, and an combination of the antibody, hIGF-II, and 50 μmol/L cytochalasin D are in green, pink, and blue, respectively.

    Techniques Used: FACS, Binding Assay, Expressing, Incubation

    23) Product Images from "Differential Roles for Actin Polymerization and a Myosin II Motor in Assembly of the Epithelial Apical Junctional Complex"

    Article Title: Differential Roles for Actin Polymerization and a Myosin II Motor in Assembly of the Epithelial Apical Junctional Complex

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E05-01-0043

    Formation of nascent junctions and TJs depends on the integrity of the actin cytoskeleton and is accompanied by global increase in intracellular F-actin. (A) In nascent junctions, E-cadherin (red) colocalizes with F-actin (green) cables that radiate between adjacent cells (arrows), whereas in TJs, occludin (red) colocalizes with a linear perijunctional F-actin belt (arrowheads). Bar, 10 μm. (B) Control T84 cells subjected to the calcium switch for either 1 or 3 h show typical adherens-like junctions and TJs at early and late stages of junctional assembly, whereas cells that were calcium-repleted for the same times in the presence of F-actin–disorganizing drug cytochalasin D (10 μM) develop neither nascent junctions nor TJs. Bar, 20 μm. (C) Representative Western blot and densitometric quantification show a dramatic decrease of G/F actin ratio starting from the early time point during calcium repletion. Data are presented as mean ± SE (n = 3); *p
    Figure Legend Snippet: Formation of nascent junctions and TJs depends on the integrity of the actin cytoskeleton and is accompanied by global increase in intracellular F-actin. (A) In nascent junctions, E-cadherin (red) colocalizes with F-actin (green) cables that radiate between adjacent cells (arrows), whereas in TJs, occludin (red) colocalizes with a linear perijunctional F-actin belt (arrowheads). Bar, 10 μm. (B) Control T84 cells subjected to the calcium switch for either 1 or 3 h show typical adherens-like junctions and TJs at early and late stages of junctional assembly, whereas cells that were calcium-repleted for the same times in the presence of F-actin–disorganizing drug cytochalasin D (10 μM) develop neither nascent junctions nor TJs. Bar, 20 μm. (C) Representative Western blot and densitometric quantification show a dramatic decrease of G/F actin ratio starting from the early time point during calcium repletion. Data are presented as mean ± SE (n = 3); *p

    Techniques Used: Western Blot

    24) Product Images from "Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes"

    Article Title: Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-017-0845-2

    No effect of MW150 treatment on BV2 cell proliferation, migration, and phagocytosis. a Average BV2 cell density at 30 h after plating in a 96-well plate at 5000 cells/well. b A representative example of the cell density at 24 h after treatment with saline, MW150 (0, 3.75, 7.5, or 15 μM), or cytochalasin D (cytD;1 μM) (mean ± SEM, n = 3 independent experiments; 8 technical replicates included for each experiment). c Quantification of pHrodo-labeled E. coli bioparticles at 3 h after addition of bioparticles. d A representative example of the cell bioparticles uptake at 3 h after treatment with saline, MW150 (0, 3.75, 7.5, or 15 μM), or cytD (1 μM) (mean ± SEM, n = 3 independent experiments; 4 technical replicates included for each experiment). e Average size of scratch wound that is filled with cells, as determined by the percent confluency in the area left nearly devoid of cells after the scratch wound, normalized to veh at 12 h post scratch. f Representative images of the scratch wound made (highlighted by green lines ), at time 0 and 12 h post scratch (mean ± SEM, n = 3 independent experiments; 8 technical replicates included for each experiment). g MW150 concentration-dependent inhibition of TNFα levels in LPS-stimulated BV2 cells (mean ± SEM, n = 1–3 independent experiments; 4 technical replicates included for each experiment). Source data is available in Additional file 4 : Table S4
    Figure Legend Snippet: No effect of MW150 treatment on BV2 cell proliferation, migration, and phagocytosis. a Average BV2 cell density at 30 h after plating in a 96-well plate at 5000 cells/well. b A representative example of the cell density at 24 h after treatment with saline, MW150 (0, 3.75, 7.5, or 15 μM), or cytochalasin D (cytD;1 μM) (mean ± SEM, n = 3 independent experiments; 8 technical replicates included for each experiment). c Quantification of pHrodo-labeled E. coli bioparticles at 3 h after addition of bioparticles. d A representative example of the cell bioparticles uptake at 3 h after treatment with saline, MW150 (0, 3.75, 7.5, or 15 μM), or cytD (1 μM) (mean ± SEM, n = 3 independent experiments; 4 technical replicates included for each experiment). e Average size of scratch wound that is filled with cells, as determined by the percent confluency in the area left nearly devoid of cells after the scratch wound, normalized to veh at 12 h post scratch. f Representative images of the scratch wound made (highlighted by green lines ), at time 0 and 12 h post scratch (mean ± SEM, n = 3 independent experiments; 8 technical replicates included for each experiment). g MW150 concentration-dependent inhibition of TNFα levels in LPS-stimulated BV2 cells (mean ± SEM, n = 1–3 independent experiments; 4 technical replicates included for each experiment). Source data is available in Additional file 4 : Table S4

    Techniques Used: Migration, Labeling, Concentration Assay, Inhibition

    25) Product Images from "HIV-1 Cell to Cell Transfer across an Env-induced, Actin-dependent Synapse"

    Article Title: HIV-1 Cell to Cell Transfer across an Env-induced, Actin-dependent Synapse

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20030648

    Receptor clustering requires the actin cytoskeleton. (A) Effector target cell conjugates were incubated at 37°C for 1 h in the presence of mAbs for CD4 (red) and Env (green). Conjugates were fixed, permeabilized, and stained for actin (blue). Clusters of f-actin on the target cell are indicated with arrows. (B) Target cells were treated with 1 μM of cytochalasin D (top) or jasplakinolide (bottom) for 1 h at 37°C. Treated cells were washed and incubated with effector cells at 37°C for 1 h with mAbs against CD4 (red) and Env (green).
    Figure Legend Snippet: Receptor clustering requires the actin cytoskeleton. (A) Effector target cell conjugates were incubated at 37°C for 1 h in the presence of mAbs for CD4 (red) and Env (green). Conjugates were fixed, permeabilized, and stained for actin (blue). Clusters of f-actin on the target cell are indicated with arrows. (B) Target cells were treated with 1 μM of cytochalasin D (top) or jasplakinolide (bottom) for 1 h at 37°C. Treated cells were washed and incubated with effector cells at 37°C for 1 h with mAbs against CD4 (red) and Env (green).

    Techniques Used: Incubation, Staining

    26) Product Images from "Unexpected link between an antibiotic, pannexin channels, and apoptosis"

    Article Title: Unexpected link between an antibiotic, pannexin channels, and apoptosis

    Journal: Nature

    doi: 10.1038/nature13147

    Formation of apoptotic bodies but not string-like apoptopodia structures is dependent on actomyosin contraction a , Time-lapse images monitoring apoptotic cell morphology of cells treated with or without CBX (500 μM) and in the presence of actomyosin contraction inhibitors. Top right, percentage of apoptotic cells forming string-like apoptopodia structures (387, 414, 459 and 372 apoptotic cells were analysed for Y-27632, Y-27632+CBX, Cyto-D and Cyto-D+CBX-treated cells, respectively, from three independent experiments). b , c , Time-lapse images monitoring apoptotic cell morphology of cells stably expressing the dominant-negative PANX1 mutant (PANX DN mutant) (b ) or treated with 40 μM trovafloxacin ( c ) in the presence of Cyto-D (5 μM). d , Inhibitors of blebbing, Y-27632, blebbistatin, or cytochalasin D (Cyto-D) reduce the formation of apoptotic bodies in Jurkat cells expressing PANX1 DN mutant ( n =3). e , Generation of apoptotic bodies by dying cells treated with Y-27632 (10 μM), blebbistatin (50 μM) and Cyto-D (5 μM). Cells were induced to undergo apoptosis in the presence or absence of CBX (500 μM) ( n =3). f , The enhanced formation of apoptotic bodies in apoptotic thymocytes from mice with PANX1 deficiency is also blunted by the ROCK inhibitor Y-27632 (10 μM) that blocks membrane blebbing ( n =3). Error bars represent s.e.m. Scale bars represent 5 μm. Arrows, apoptopodia.
    Figure Legend Snippet: Formation of apoptotic bodies but not string-like apoptopodia structures is dependent on actomyosin contraction a , Time-lapse images monitoring apoptotic cell morphology of cells treated with or without CBX (500 μM) and in the presence of actomyosin contraction inhibitors. Top right, percentage of apoptotic cells forming string-like apoptopodia structures (387, 414, 459 and 372 apoptotic cells were analysed for Y-27632, Y-27632+CBX, Cyto-D and Cyto-D+CBX-treated cells, respectively, from three independent experiments). b , c , Time-lapse images monitoring apoptotic cell morphology of cells stably expressing the dominant-negative PANX1 mutant (PANX DN mutant) (b ) or treated with 40 μM trovafloxacin ( c ) in the presence of Cyto-D (5 μM). d , Inhibitors of blebbing, Y-27632, blebbistatin, or cytochalasin D (Cyto-D) reduce the formation of apoptotic bodies in Jurkat cells expressing PANX1 DN mutant ( n =3). e , Generation of apoptotic bodies by dying cells treated with Y-27632 (10 μM), blebbistatin (50 μM) and Cyto-D (5 μM). Cells were induced to undergo apoptosis in the presence or absence of CBX (500 μM) ( n =3). f , The enhanced formation of apoptotic bodies in apoptotic thymocytes from mice with PANX1 deficiency is also blunted by the ROCK inhibitor Y-27632 (10 μM) that blocks membrane blebbing ( n =3). Error bars represent s.e.m. Scale bars represent 5 μm. Arrows, apoptopodia.

    Techniques Used: Stable Transfection, Expressing, Dominant Negative Mutation, Mutagenesis, Mouse Assay

    27) Product Images from "Cell adhesion-mediated mitochondria transfer contributes to mesenchymal stem cell-induced chemoresistance on T cell acute lymphoblastic leukemia cells"

    Article Title: Cell adhesion-mediated mitochondria transfer contributes to mesenchymal stem cell-induced chemoresistance on T cell acute lymphoblastic leukemia cells

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-018-0554-z

    Inhibition of TNT formation ameliorates MSC-induced chemoresistance on primary T-ALL cells. Cytochalasin D (1 μM) was added to the coculture system with ara-C or MTX for 48 h. a Representative photos of human primary T-ALL cells and MSCs in the coculture system after the removal of nonadhesive human primary T-ALL cells. Scale bar, 100 μm. b Representative confocal microscopy images show that human primary T-ALL cell-derived mitochondria (Red+) were internalized in MSCs. Scale bar, 20 μm. The areas of red foci per field were calculated by ImageJ software. c The apoptosis rate was determined using annexin V/PI staining and FACS. d The percentages of annexin V-positive cells were calculated. e A CCK-8 assay was used to assess human primary T-ALL cell viability. f Graphic abstract: T-ALL cell/MSC adhesion-mediated mitochondria transfer contributes to MSC-induced chemoresistance. The data above are presented as the mean ± S.E.M. of three independent experiments (* P
    Figure Legend Snippet: Inhibition of TNT formation ameliorates MSC-induced chemoresistance on primary T-ALL cells. Cytochalasin D (1 μM) was added to the coculture system with ara-C or MTX for 48 h. a Representative photos of human primary T-ALL cells and MSCs in the coculture system after the removal of nonadhesive human primary T-ALL cells. Scale bar, 100 μm. b Representative confocal microscopy images show that human primary T-ALL cell-derived mitochondria (Red+) were internalized in MSCs. Scale bar, 20 μm. The areas of red foci per field were calculated by ImageJ software. c The apoptosis rate was determined using annexin V/PI staining and FACS. d The percentages of annexin V-positive cells were calculated. e A CCK-8 assay was used to assess human primary T-ALL cell viability. f Graphic abstract: T-ALL cell/MSC adhesion-mediated mitochondria transfer contributes to MSC-induced chemoresistance. The data above are presented as the mean ± S.E.M. of three independent experiments (* P

    Techniques Used: Inhibition, Acetylene Reduction Assay, Confocal Microscopy, Derivative Assay, Software, Staining, FACS, CCK-8 Assay

    Mitochondria transfer from Jurkat cells to MSCs can be blocked by cytochalasin D. a Representative confocal microscopy images show the presence of TNTs containing mitochondria (arrow). Scale bar, 5 μm. b 18-α-GA (50 μM), dynasore (50 μM), or cytochalasin D (1 μM) was added to the coculture system with ara-C or MTX for 48 h. Flow cytometry analysis of Jurkat cell-derived mitochondria uptake by MSCs (GFP+ gated). c The percentage of Red+ MSCs in each group was analyzed and graphed. The results are expressed as the mean ± S.E.M. of three independent experiments (* P
    Figure Legend Snippet: Mitochondria transfer from Jurkat cells to MSCs can be blocked by cytochalasin D. a Representative confocal microscopy images show the presence of TNTs containing mitochondria (arrow). Scale bar, 5 μm. b 18-α-GA (50 μM), dynasore (50 μM), or cytochalasin D (1 μM) was added to the coculture system with ara-C or MTX for 48 h. Flow cytometry analysis of Jurkat cell-derived mitochondria uptake by MSCs (GFP+ gated). c The percentage of Red+ MSCs in each group was analyzed and graphed. The results are expressed as the mean ± S.E.M. of three independent experiments (* P

    Techniques Used: Confocal Microscopy, Acetylene Reduction Assay, Flow Cytometry, Cytometry, Derivative Assay

    Inhibition of mitochondria transfer decreases the effects of MSC-induced chemoresistance. a , b Cytochalasin D (1 μM) was added to the coculture system with ara-C or MTX for 48 h. The levels of mitochondrial ROS in Jurkat cells were examined by MitoSOX staining. c The apoptosis rate was determined using annexin V/PI staining and FACS. d The percentages of annexin V-positive cells were calculated. e A CCK-8 assay was used to assess Jurkat cell viability. The data above are presented as the mean ± S.E.M. of three independent experiments (* P
    Figure Legend Snippet: Inhibition of mitochondria transfer decreases the effects of MSC-induced chemoresistance. a , b Cytochalasin D (1 μM) was added to the coculture system with ara-C or MTX for 48 h. The levels of mitochondrial ROS in Jurkat cells were examined by MitoSOX staining. c The apoptosis rate was determined using annexin V/PI staining and FACS. d The percentages of annexin V-positive cells were calculated. e A CCK-8 assay was used to assess Jurkat cell viability. The data above are presented as the mean ± S.E.M. of three independent experiments (* P

    Techniques Used: Inhibition, Acetylene Reduction Assay, Staining, FACS, CCK-8 Assay

    28) Product Images from "Endocytic uptake of monomeric amyloid-β peptides is clathrin- and dynamin-independent and results in selective accumulation of Aβ(1–42) compared to Aβ(1–40)"

    Article Title: Endocytic uptake of monomeric amyloid-β peptides is clathrin- and dynamin-independent and results in selective accumulation of Aβ(1–42) compared to Aβ(1–40)

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02227-9

    Uptake of Aβ(1–40), Aβ(1–42) and Trf in SH-SY5Y cells under conditions that perturb actin dependent endocytosis, macropinocytosis and Arf6-dependent endocytosis. ( a ) Uptake of HF488-labelled Aβ(1–40), Aβ(1–42) and AF647-labelled Trf in cells treated with 10 µM cytochalasin A (cyto A) and 25 µg/ml cytochalasin D (cyto D) to perturb actin polymerisation, or 10 µM IPA-3 and 25 nM wortmannin (wort) to perturb macropinocytosis. The peptide uptake is reported as mean cellular uptake relative to control (cells not treated with inhibitor) calculated based on mean cellular fluorescence intensity ± SD of the total number of gated live cells for three replicate samples (n = 3) measured by flow cytometry and corrected for baseline contributions by subtracting the cellular autofluorescence. ( b , c ) Images showing uptake of HF488-labelled Aβ(1–40) ( b ) and Aβ(1–42) ( c ) (green) in cells transfected with BFP-labelled Arf6 WT or Q67L (blue) alongside transmitted images showing the cells. The cells were incubated with Aβ 24 h post transfection. The Aβ concentration was 1 μM and the concentration of Trf was 5 µg/ml in all experiments. Cells were incubated with Aβ for 1 h (flow cytometry) or 24 h (confocal microscopy), and Trf for 5 min.
    Figure Legend Snippet: Uptake of Aβ(1–40), Aβ(1–42) and Trf in SH-SY5Y cells under conditions that perturb actin dependent endocytosis, macropinocytosis and Arf6-dependent endocytosis. ( a ) Uptake of HF488-labelled Aβ(1–40), Aβ(1–42) and AF647-labelled Trf in cells treated with 10 µM cytochalasin A (cyto A) and 25 µg/ml cytochalasin D (cyto D) to perturb actin polymerisation, or 10 µM IPA-3 and 25 nM wortmannin (wort) to perturb macropinocytosis. The peptide uptake is reported as mean cellular uptake relative to control (cells not treated with inhibitor) calculated based on mean cellular fluorescence intensity ± SD of the total number of gated live cells for three replicate samples (n = 3) measured by flow cytometry and corrected for baseline contributions by subtracting the cellular autofluorescence. ( b , c ) Images showing uptake of HF488-labelled Aβ(1–40) ( b ) and Aβ(1–42) ( c ) (green) in cells transfected with BFP-labelled Arf6 WT or Q67L (blue) alongside transmitted images showing the cells. The cells were incubated with Aβ 24 h post transfection. The Aβ concentration was 1 μM and the concentration of Trf was 5 µg/ml in all experiments. Cells were incubated with Aβ for 1 h (flow cytometry) or 24 h (confocal microscopy), and Trf for 5 min.

    Techniques Used: Indirect Immunoperoxidase Assay, Fluorescence, Flow Cytometry, Cytometry, Transfection, Incubation, Concentration Assay, Confocal Microscopy

    29) Product Images from "Phagocytosis of environmental or metabolic crystalline particles induces cytotoxicity by triggering necroptosis across a broad range of particle size and shape"

    Article Title: Phagocytosis of environmental or metabolic crystalline particles induces cytotoxicity by triggering necroptosis across a broad range of particle size and shape

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-15804-9

    Phagocytosis of crystals or crystalline particles triggers their cytotoxicity. HK-2 cells were pretreated with the phagocytosis inhibitor cytochalasin D (Cyt D) (10 µM) 30 min before exposing to CaP (1 mg/ml), silica (1 mg/ml), TiO 2 (0.5 mg/ml), cholesterol (3 mg/ml), CaOx (1 mg/ml), and MSU (0.5 mg/ml) for 24 hrs. ( A ) Cell death was visualized by PI stain (red color) and Acridine orange (green color). PI images were converted into black and white image for better visualization using ImageJ software. ( B ) Quantification of DNA-PI mean florescence intensity (MFI). Data are expressed as mean ± SEM from three independent experiments.
    Figure Legend Snippet: Phagocytosis of crystals or crystalline particles triggers their cytotoxicity. HK-2 cells were pretreated with the phagocytosis inhibitor cytochalasin D (Cyt D) (10 µM) 30 min before exposing to CaP (1 mg/ml), silica (1 mg/ml), TiO 2 (0.5 mg/ml), cholesterol (3 mg/ml), CaOx (1 mg/ml), and MSU (0.5 mg/ml) for 24 hrs. ( A ) Cell death was visualized by PI stain (red color) and Acridine orange (green color). PI images were converted into black and white image for better visualization using ImageJ software. ( B ) Quantification of DNA-PI mean florescence intensity (MFI). Data are expressed as mean ± SEM from three independent experiments.

    Techniques Used: Staining, Software

    Schematic representation of molecular mechanisms of crystalline particle-induced necroptosis. After exposure, crystals or crystalline particles are phagocytized by human cells. The frustrated crystal phagocytosis leads to lysosomal destabilization and subsequent leakage of cathepsin B (or other lytic proteases) into the cytosol. This cytosolic cathepsin B binds directly to RIPK1, an indigenous inhibitor of necroptosis, and induces its degradation. The degradation of RIPK1 leads to the formation of the necrosome that consists of RIPK3 and MLKL. A series of auto/trans phosphorylation activate RIPK3 and MLKL, leading to formation of oligomers of MLKL, which then are translocated to different membranes, e.g. nucleus, endoplasmic reticulum, mitochondria and plasma membranes etc. After binding MLKL, oligomers induce pore formation in these membranes leading to necroptosis. Crystals or crystalline particle-induced necroptosis can be inhibited by blocking phagocytosis using cytochalasin D or necroptosis using RIPK1 inhibitors necrostatin-1s, RIPK3 inhibitor dabrafenib, and MLKL inhibitor necrosulfonamide. Cat-B: Cathepsin B, RIPK: Receptor interacting protein kinase, MLKL: Mixed lineage kinase domain-like.
    Figure Legend Snippet: Schematic representation of molecular mechanisms of crystalline particle-induced necroptosis. After exposure, crystals or crystalline particles are phagocytized by human cells. The frustrated crystal phagocytosis leads to lysosomal destabilization and subsequent leakage of cathepsin B (or other lytic proteases) into the cytosol. This cytosolic cathepsin B binds directly to RIPK1, an indigenous inhibitor of necroptosis, and induces its degradation. The degradation of RIPK1 leads to the formation of the necrosome that consists of RIPK3 and MLKL. A series of auto/trans phosphorylation activate RIPK3 and MLKL, leading to formation of oligomers of MLKL, which then are translocated to different membranes, e.g. nucleus, endoplasmic reticulum, mitochondria and plasma membranes etc. After binding MLKL, oligomers induce pore formation in these membranes leading to necroptosis. Crystals or crystalline particle-induced necroptosis can be inhibited by blocking phagocytosis using cytochalasin D or necroptosis using RIPK1 inhibitors necrostatin-1s, RIPK3 inhibitor dabrafenib, and MLKL inhibitor necrosulfonamide. Cat-B: Cathepsin B, RIPK: Receptor interacting protein kinase, MLKL: Mixed lineage kinase domain-like.

    Techniques Used: Binding Assay, Blocking Assay

    30) Product Images from "PKCα diffusion and translocation are independent of an intact cytoskeleton"

    Article Title: PKCα diffusion and translocation are independent of an intact cytoskeleton

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00560-7

    Depolymerization of actin filaments and microtubules does not alter Ca 2+ induced PKCα translocation. ( A ) Cytochalasin-D (4 µM, 2 hours) (b,f) or nocodazole (10 µM, 2 hours) (d,h) treatment of HEK cells, either expressing Lifeact-GFP (a,b) and α-tubulin-eGFP (c,d), staining with phalloidin (e,f) or immunofluorescence with primary antibodies against α-tubulin (g,h), results in depolymerisation of actin filaments (b,f) and microtubules (d,h), respectively. Note that the confocal sections in Aa and Ab were deliberately close to the bottom of the cell to highlight the spiky plasma membrane protrusion while all other confocal sections were placed in the middle of the nucleus. ( B ) Exemplified single images of PKCα-eYFP distribution before (Ba1, Bb1, Bc1) and following (Ba2, Bb2, Bc2) photolytic Ca 2+ increase at the time points marked in the traces to the right. Three different experimental conditions are depicted (Ba-control, Bb-cytochalasin-D treatment, Bc- nocodazole treatment). Traces were generated from regions of interest in the cytosol (red traces) and on the plasma membrane (blue traces). ( C ) The plasma membrane accumulation was characterized by fitting an exponential to the upstroke following the flash photolytic Ca 2+ increase. The statistical summary demonstrates that the state of polymerization of the cytoskeleton does not influence the speed at which PKCα-eYFP accumulates at the plasma membrane after Ca 2+ UV flash photolysis. Numbers given on the bars indicate number of cells in at least 5 independent experiments.
    Figure Legend Snippet: Depolymerization of actin filaments and microtubules does not alter Ca 2+ induced PKCα translocation. ( A ) Cytochalasin-D (4 µM, 2 hours) (b,f) or nocodazole (10 µM, 2 hours) (d,h) treatment of HEK cells, either expressing Lifeact-GFP (a,b) and α-tubulin-eGFP (c,d), staining with phalloidin (e,f) or immunofluorescence with primary antibodies against α-tubulin (g,h), results in depolymerisation of actin filaments (b,f) and microtubules (d,h), respectively. Note that the confocal sections in Aa and Ab were deliberately close to the bottom of the cell to highlight the spiky plasma membrane protrusion while all other confocal sections were placed in the middle of the nucleus. ( B ) Exemplified single images of PKCα-eYFP distribution before (Ba1, Bb1, Bc1) and following (Ba2, Bb2, Bc2) photolytic Ca 2+ increase at the time points marked in the traces to the right. Three different experimental conditions are depicted (Ba-control, Bb-cytochalasin-D treatment, Bc- nocodazole treatment). Traces were generated from regions of interest in the cytosol (red traces) and on the plasma membrane (blue traces). ( C ) The plasma membrane accumulation was characterized by fitting an exponential to the upstroke following the flash photolytic Ca 2+ increase. The statistical summary demonstrates that the state of polymerization of the cytoskeleton does not influence the speed at which PKCα-eYFP accumulates at the plasma membrane after Ca 2+ UV flash photolysis. Numbers given on the bars indicate number of cells in at least 5 independent experiments.

    Techniques Used: Translocation Assay, Expressing, Staining, Immunofluorescence, Generated

    31) Product Images from "Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8"

    Article Title: Agonist-Dependent Modulation of Cell Surface Expression of the Cold Receptor TRPM8

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3820-13.2015

    TRPM8 internalization and recruitment are affected after menthol. A , Distribution of particles follow a two-state model, where equilibrium constant values during exocytic and endocytic processes vary in the presence of menthol. B , Partial inhibition of endocytosis processes after MβCD incubation. Decay constant values (τ) indicate that menthol stimulates MβCD-dependent exocytosis by ∼6 times (blue circles) in comparison with control conditions (gray circles). Purple shaded regions on the TIRF images represent higher intensity, suggesting an increase in TRPM8-containing vesicles at PM. C , Partial inhibition of exocytosis processes induced by cytoD incubation. Top, Images represent overall presence of particles after cytoD incubation. Purple shaded regions represent higher intensity, suggesting a decrease in the exocytic rate. Comparison of decay constant values (τ) shows that menthol has a near twofold effect on cytoD-dependent vesicle endocytosis. n = 6.
    Figure Legend Snippet: TRPM8 internalization and recruitment are affected after menthol. A , Distribution of particles follow a two-state model, where equilibrium constant values during exocytic and endocytic processes vary in the presence of menthol. B , Partial inhibition of endocytosis processes after MβCD incubation. Decay constant values (τ) indicate that menthol stimulates MβCD-dependent exocytosis by ∼6 times (blue circles) in comparison with control conditions (gray circles). Purple shaded regions on the TIRF images represent higher intensity, suggesting an increase in TRPM8-containing vesicles at PM. C , Partial inhibition of exocytosis processes induced by cytoD incubation. Top, Images represent overall presence of particles after cytoD incubation. Purple shaded regions represent higher intensity, suggesting a decrease in the exocytic rate. Comparison of decay constant values (τ) shows that menthol has a near twofold effect on cytoD-dependent vesicle endocytosis. n = 6.

    Techniques Used: Inhibition, Incubation

    32) Product Images from "Inter-subunit interactions drive divergent dynamics in mammalian and Plasmodium actin filaments"

    Article Title: Inter-subunit interactions drive divergent dynamics in mammalian and Plasmodium actin filaments

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2005345

    Coronin displays reduced binding, yet overexpression rescues the PbS4Oc phenotype. (A) Overexpression of actin binding proteins specifically in the sporozoite stage of the life cycle. (B) Coronin localises at the back of motile wild-type but along the cell periphery in PbS4Oc sporozoites or sporozoites treated with CD, suggesting reduced efficiency of coronin binding. Red arrowheads indicate the front of the sporozoites, scale bar: 5 μm. (C–E) Profilin and ADF2 overexpression were unable to restore organ penetration (c), motility (d), and reduce pausing (e), while coronin overexpression was able to partially rescue the phenotype observed in the PbS4Oc line. In (C) data represented by mean ± standard error. In (D) black, grey, and white bars: efficiently, partially, and non-motile but attached sporozoites, respectively. Fisher’s exact test comparing moving groups in [D] and 0 pause groups in [E];  n  is the total numbers of parasites counted per group. A coronin actin binding mutant (K349E,R350E) and a coronin construct lacking the C-terminal region (yet having the intact actin binding domain, ΔC) were unable to rescue the phenotype, suggesting an interplay between N- and C-terminal regions to mediate rescue. Please note that the wild-type and PbS4Oc data are the same as from   Fig 2C, 2F and 2H . Underlying data can be found in   S1 Data . ADF2, actin depolymerising factor 2; CD, cytochalasin D; ΔC, coronin construct lacking C-terminal region; K349E,R350E, full length coronin actin binding mutant; mCh, mCherry.
    Figure Legend Snippet: Coronin displays reduced binding, yet overexpression rescues the PbS4Oc phenotype. (A) Overexpression of actin binding proteins specifically in the sporozoite stage of the life cycle. (B) Coronin localises at the back of motile wild-type but along the cell periphery in PbS4Oc sporozoites or sporozoites treated with CD, suggesting reduced efficiency of coronin binding. Red arrowheads indicate the front of the sporozoites, scale bar: 5 μm. (C–E) Profilin and ADF2 overexpression were unable to restore organ penetration (c), motility (d), and reduce pausing (e), while coronin overexpression was able to partially rescue the phenotype observed in the PbS4Oc line. In (C) data represented by mean ± standard error. In (D) black, grey, and white bars: efficiently, partially, and non-motile but attached sporozoites, respectively. Fisher’s exact test comparing moving groups in [D] and 0 pause groups in [E]; n is the total numbers of parasites counted per group. A coronin actin binding mutant (K349E,R350E) and a coronin construct lacking the C-terminal region (yet having the intact actin binding domain, ΔC) were unable to rescue the phenotype, suggesting an interplay between N- and C-terminal regions to mediate rescue. Please note that the wild-type and PbS4Oc data are the same as from Fig 2C, 2F and 2H . Underlying data can be found in S1 Data . ADF2, actin depolymerising factor 2; CD, cytochalasin D; ΔC, coronin construct lacking C-terminal region; K349E,R350E, full length coronin actin binding mutant; mCh, mCherry.

    Techniques Used: Binding Assay, Over Expression, Mutagenesis, Construct

    33) Product Images from "Mapping Organelle Motion Reveals a Vesicular Conveyor Belt Spatially Replenishing Secretory Vesicles in Stimulated Chromaffin Cells"

    Article Title: Mapping Organelle Motion Reveals a Vesicular Conveyor Belt Spatially Replenishing Secretory Vesicles in Stimulated Chromaffin Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087242

    Blocking actin and microtubule polymerization interferes with the replenishment mechanism. ( A, B, C ) Chromaffin cells were incubated with cytochalasin D (10 µM) for 20 minutes before imaging and stimulation. ( A ) Comparison of percentages of vesicles lying in the three different pools in control conditions and during nicotine stimulation (10 µM) (N = 5 cells, n = 978 vesicles). ( B ) Histogram of accumulated vesicle positions depending on their motion and distance from the membrane. ( C ) Data from stimulated cells were used to generate maps of the average percentage of vesicles in a given motion state, relative to their distance from the plasma membrane. For analysis purposes, the cells have been divided in 0.5 µm zones. The central part of cells is not represented due to insufficient data and uncertainties regarding the closest membrane direction. ( D , E , F ) Chromaffin cells were incubated with nocodazole (20 µM) for 20 minutes before imaging and nicotine stimulation. ( D ) Comparison of percentage of vesicles lying in the three different pools in control conditions and during stimulation of exocytosis following nicotine (10 µM) stimulation (N = 4 cells, n = 629 vesicles). ( E ) Histogram of accumulated vesicle positions depending on their motion and distance from the membrane. ( F ) Data from stimulated cells were used to generate maps of the average percentage of vesicles in a given motion state, based on their distance from the plasma membrane.
    Figure Legend Snippet: Blocking actin and microtubule polymerization interferes with the replenishment mechanism. ( A, B, C ) Chromaffin cells were incubated with cytochalasin D (10 µM) for 20 minutes before imaging and stimulation. ( A ) Comparison of percentages of vesicles lying in the three different pools in control conditions and during nicotine stimulation (10 µM) (N = 5 cells, n = 978 vesicles). ( B ) Histogram of accumulated vesicle positions depending on their motion and distance from the membrane. ( C ) Data from stimulated cells were used to generate maps of the average percentage of vesicles in a given motion state, relative to their distance from the plasma membrane. For analysis purposes, the cells have been divided in 0.5 µm zones. The central part of cells is not represented due to insufficient data and uncertainties regarding the closest membrane direction. ( D , E , F ) Chromaffin cells were incubated with nocodazole (20 µM) for 20 minutes before imaging and nicotine stimulation. ( D ) Comparison of percentage of vesicles lying in the three different pools in control conditions and during stimulation of exocytosis following nicotine (10 µM) stimulation (N = 4 cells, n = 629 vesicles). ( E ) Histogram of accumulated vesicle positions depending on their motion and distance from the membrane. ( F ) Data from stimulated cells were used to generate maps of the average percentage of vesicles in a given motion state, based on their distance from the plasma membrane.

    Techniques Used: Blocking Assay, Incubation, Imaging

    34) Product Images from "Epithelial Monolayers Coalesce on a Viscoelastic Substrate through Redistribution of Vinculin"

    Article Title: Epithelial Monolayers Coalesce on a Viscoelastic Substrate through Redistribution of Vinculin

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2017.07.027

    Coalescence is regulated by FAK-mediated actomyosin contractility. ( A ) Shown here is PIV analysis of cell movement in CL-S1 cells on VE PDMS. ( B ) CL-S1 monolayers on VE PDMS were treated with cytochalasin D ( cytoD ), blebbistatin ( Bleb ), and RhoK inhibitor Y27632, and the cell-free area was measured at 5 h postsettling ( n = 5–8). ( C ) CL-S1 and MDCK monolayers on PDMS substrata were treated with 2 nM Calyculin A ( CalA ) or control DMSO for 2 h ( n = 5–8). ( D ) CL-S1 monolayers on VE PDMS were treated with FAK inhibitor PF-562271 (FAK inh) and the cell-free area was measured 6 h postsettling ( n = 3–4). ( E ) CL-S1 monolayers on VE PDMS were treated with control DMSO, 2 nM Calyculin A ( CalA ), or 2 nM CalA + 10 nM FAK inhibitor PF-562271 ( CalA + FAK inh ) ( n = 3). ( F ) Given here are confocal images of CL-S1 cells on VE PDMS fixed and immunostained 1 h postsettling. ( F′ ) Shown here is quantitative image analysis on the levels of p-FAK relative to FAK (FAK pixels colocalized with pFAK/total FAK pixels) across different PDMS substrata ( F′ , n = 6–9). ( G ) Shown here is Western blot of whole cell lysates of CL-S1 monolayers on different PDMS substrata 1 h postsettling, immunoblotted for FAK and phospho-FAK Tyr 397 ( p-FAK ) and β -actin. ( H ) Given here are confocal projections of CL-S1 cells on different PDMS substrata fixed and immunostained for MCL/pMCL, F-actin, and nucleus, 4 h postsettling. ( I ) Confocal projections of CL-S1 cells are shown on different PDMS substrata fixed and immunostained for MyoIIA/B, F-actin, and nucleus, 4 h postsettling. ∗ p
    Figure Legend Snippet: Coalescence is regulated by FAK-mediated actomyosin contractility. ( A ) Shown here is PIV analysis of cell movement in CL-S1 cells on VE PDMS. ( B ) CL-S1 monolayers on VE PDMS were treated with cytochalasin D ( cytoD ), blebbistatin ( Bleb ), and RhoK inhibitor Y27632, and the cell-free area was measured at 5 h postsettling ( n = 5–8). ( C ) CL-S1 and MDCK monolayers on PDMS substrata were treated with 2 nM Calyculin A ( CalA ) or control DMSO for 2 h ( n = 5–8). ( D ) CL-S1 monolayers on VE PDMS were treated with FAK inhibitor PF-562271 (FAK inh) and the cell-free area was measured 6 h postsettling ( n = 3–4). ( E ) CL-S1 monolayers on VE PDMS were treated with control DMSO, 2 nM Calyculin A ( CalA ), or 2 nM CalA + 10 nM FAK inhibitor PF-562271 ( CalA + FAK inh ) ( n = 3). ( F ) Given here are confocal images of CL-S1 cells on VE PDMS fixed and immunostained 1 h postsettling. ( F′ ) Shown here is quantitative image analysis on the levels of p-FAK relative to FAK (FAK pixels colocalized with pFAK/total FAK pixels) across different PDMS substrata ( F′ , n = 6–9). ( G ) Shown here is Western blot of whole cell lysates of CL-S1 monolayers on different PDMS substrata 1 h postsettling, immunoblotted for FAK and phospho-FAK Tyr 397 ( p-FAK ) and β -actin. ( H ) Given here are confocal projections of CL-S1 cells on different PDMS substrata fixed and immunostained for MCL/pMCL, F-actin, and nucleus, 4 h postsettling. ( I ) Confocal projections of CL-S1 cells are shown on different PDMS substrata fixed and immunostained for MyoIIA/B, F-actin, and nucleus, 4 h postsettling. ∗ p

    Techniques Used: Western Blot

    35) Product Images from "The ocular albinism type 1 protein, an intracellular G protein-coupled receptor, regulates melanosome transport in pigment cells"

    Article Title: The ocular albinism type 1 protein, an intracellular G protein-coupled receptor, regulates melanosome transport in pigment cells

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddn241

    Melanosomes redistribute according to MT density towards the nucleus upon disruption of the actin network in wild-type and Oa1 -KO melanocytes. ( A ) Melanosome distribution and cytoskeletal organization. Pictures show the typical distribution of melanosomes, visualized in bright field, compared with tubulin or actin filaments, visualized by indirect immunofluorescence with anti-tubulin antibodies or phalloidin staining, respectively, in Oa1 +/− and Oa1 −/− cells. Black/white circles indicate the perinuclear area, as defined in organelle tracking analyses (see Materials and Methods), which is typically enriched in MTs (and melanosomes in wild-type, but not mutant, cells), while AF are more abundant at the cell periphery. Arrowheads point to the position of the centrosome. In Oa1 −/− melanocytes, despite a similar cytoskeletal organization, melanosomes appear excluded from the MT-enriched perinuclear region. ( B ) Melanosome redistribution upon disruption of the actin cytoskeleton. Shown are representative bright field pictures of Oa1 +/− and Oa1 −/− cells prior to treatment (untreated); after 1 h of cytochalasin D treatment (cyto D 1 h); and, following removal of the drug and extensive washing, allowed to recover for 1 h (recovery 1 h). In both cell types, melanosome similarly redistribute upon AF disruption and recover to the original distribution after withdrawal of the drug. ( C ) Melanosome redistribution after 1 h of cytochalasin D treatment is compared with the residual AF labelling by phalloidin under the plasma membrane, confirming the absence of generalized retraction of cell margins. Bars, 15 µm.
    Figure Legend Snippet: Melanosomes redistribute according to MT density towards the nucleus upon disruption of the actin network in wild-type and Oa1 -KO melanocytes. ( A ) Melanosome distribution and cytoskeletal organization. Pictures show the typical distribution of melanosomes, visualized in bright field, compared with tubulin or actin filaments, visualized by indirect immunofluorescence with anti-tubulin antibodies or phalloidin staining, respectively, in Oa1 +/− and Oa1 −/− cells. Black/white circles indicate the perinuclear area, as defined in organelle tracking analyses (see Materials and Methods), which is typically enriched in MTs (and melanosomes in wild-type, but not mutant, cells), while AF are more abundant at the cell periphery. Arrowheads point to the position of the centrosome. In Oa1 −/− melanocytes, despite a similar cytoskeletal organization, melanosomes appear excluded from the MT-enriched perinuclear region. ( B ) Melanosome redistribution upon disruption of the actin cytoskeleton. Shown are representative bright field pictures of Oa1 +/− and Oa1 −/− cells prior to treatment (untreated); after 1 h of cytochalasin D treatment (cyto D 1 h); and, following removal of the drug and extensive washing, allowed to recover for 1 h (recovery 1 h). In both cell types, melanosome similarly redistribute upon AF disruption and recover to the original distribution after withdrawal of the drug. ( C ) Melanosome redistribution after 1 h of cytochalasin D treatment is compared with the residual AF labelling by phalloidin under the plasma membrane, confirming the absence of generalized retraction of cell margins. Bars, 15 µm.

    Techniques Used: Immunofluorescence, Staining, Mutagenesis

    Quantification of melanosome motility in wild-type and Oa1 -KO melanocytes. ( A ) Speed distribution profile obtained by pooling and binning in increasing speed intervals, as indicated, all melanosome steps from Oa1 +/− melanocytes, or from Oa1 −/− melanocytes. Cells were analyzed either untreated and selected inside or outside of the perinuclear area (NT in and out, respectively); or after treatment with nocodazole to deplete the MTs; or following expression of the dominant-negative myosin Va construct (EGFP-MC-LT), or treatment with cytochalasin D (Cyto D) to disrupt AF-based transport. Number of melanosomes tracked: NT in = 103, NT out = 100, nocodazole = 90, EGFP-MC-LT and Cyto D = 50 (obtained from several different cells: 10–12 for NT and nocodazole; 5–7 for EGFP-MC-LT and Cyto D). Data are expressed as percent of total steps. In both melanocyte lines, speeds ≥0.4 μm/s (dark grey) are totally abolished by nocodazole, indicating that they represent a pure MT-dependent component. Oa1 −/− melanocytes show a decreased motility in untreated conditions, yet recover completely upon disruption of AF-based transport. ( B ) Average total path of melanosomes from the same tracking analysis described above for Oa1 +/− versus Oa1 −/− melanocytes, and from similar analyses performed in Oa1 +/+ versus Oa1 −/− melanocytes, and in transduced Oa1 −/− LOA1SN versus Oa1 −/− LOA1Δ18SN lines. For each wild-type and Oa1 -deficient lines compared in the graphs, tracked melanosomes were of comparable size (see Supplementary Material, Table S1 ). Results represent the mean ± SEM of the data from 40–100 melanosomes (considering one melanosome path as one data point). Compared with their wild-type counterpart, Oa1 -deficient melanocytes show a significantly reduced average displacement in untreated conditions, while they completely recover upon pEGFP-MC-LT transfection, or cytochalasin D treatment. Transduced lines showed poor motility at the cell periphery (possibly due to the drug treatment necessary for maintenance of the transgenes), so differences were only appreciable in the perinuclear area. * P
    Figure Legend Snippet: Quantification of melanosome motility in wild-type and Oa1 -KO melanocytes. ( A ) Speed distribution profile obtained by pooling and binning in increasing speed intervals, as indicated, all melanosome steps from Oa1 +/− melanocytes, or from Oa1 −/− melanocytes. Cells were analyzed either untreated and selected inside or outside of the perinuclear area (NT in and out, respectively); or after treatment with nocodazole to deplete the MTs; or following expression of the dominant-negative myosin Va construct (EGFP-MC-LT), or treatment with cytochalasin D (Cyto D) to disrupt AF-based transport. Number of melanosomes tracked: NT in = 103, NT out = 100, nocodazole = 90, EGFP-MC-LT and Cyto D = 50 (obtained from several different cells: 10–12 for NT and nocodazole; 5–7 for EGFP-MC-LT and Cyto D). Data are expressed as percent of total steps. In both melanocyte lines, speeds ≥0.4 μm/s (dark grey) are totally abolished by nocodazole, indicating that they represent a pure MT-dependent component. Oa1 −/− melanocytes show a decreased motility in untreated conditions, yet recover completely upon disruption of AF-based transport. ( B ) Average total path of melanosomes from the same tracking analysis described above for Oa1 +/− versus Oa1 −/− melanocytes, and from similar analyses performed in Oa1 +/+ versus Oa1 −/− melanocytes, and in transduced Oa1 −/− LOA1SN versus Oa1 −/− LOA1Δ18SN lines. For each wild-type and Oa1 -deficient lines compared in the graphs, tracked melanosomes were of comparable size (see Supplementary Material, Table S1 ). Results represent the mean ± SEM of the data from 40–100 melanosomes (considering one melanosome path as one data point). Compared with their wild-type counterpart, Oa1 -deficient melanocytes show a significantly reduced average displacement in untreated conditions, while they completely recover upon pEGFP-MC-LT transfection, or cytochalasin D treatment. Transduced lines showed poor motility at the cell periphery (possibly due to the drug treatment necessary for maintenance of the transgenes), so differences were only appreciable in the perinuclear area. * P

    Techniques Used: Expressing, Dominant Negative Mutation, Construct, Transfection

    36) Product Images from "A Defect in Influenza A Virus Particle Assembly Specific to Primary Human Macrophages"

    Article Title: A Defect in Influenza A Virus Particle Assembly Specific to Primary Human Macrophages

    Journal: mBio

    doi: 10.1128/mBio.01916-18

    Cytochalasin D treatment restores HA-M2 PLA in MDM to levels comparable to that in dTHP1 cells. dTHP1 cells and MDM were infected with WSN at MOI 0.1. At 14 hpi, cells were treated with vehicle control (DMSO) or 20 μM cytochalasin D (Cyto D) for 2 h before fixation. (A) Cells were analyzed as in Fig. 5A . Representative maximum intensity projection images are shown. (B) Number of PLA spots was counted for each cell. These experiments were performed in parallel with the experiments shown in Fig. S4 using MDM from the same donors. Data are from at least three independent experiments, and 8 to 10 cells were analyzed per experiment. Error bars represent standard error of mean. ****, P
    Figure Legend Snippet: Cytochalasin D treatment restores HA-M2 PLA in MDM to levels comparable to that in dTHP1 cells. dTHP1 cells and MDM were infected with WSN at MOI 0.1. At 14 hpi, cells were treated with vehicle control (DMSO) or 20 μM cytochalasin D (Cyto D) for 2 h before fixation. (A) Cells were analyzed as in Fig. 5A . Representative maximum intensity projection images are shown. (B) Number of PLA spots was counted for each cell. These experiments were performed in parallel with the experiments shown in Fig. S4 using MDM from the same donors. Data are from at least three independent experiments, and 8 to 10 cells were analyzed per experiment. Error bars represent standard error of mean. ****, P

    Techniques Used: Proximity Ligation Assay, Infection

    Cytochalasin D treatment increases bud formation in MDM to levels comparable to that in dTHP1 cells. dTHP1 cells and MDM were infected with WSN at MOI 0.1. At 14 hpi, cells were treated with vehicle control (DMSO) or 20 μM Cyto D for 4 h before fixation and immunostaining with anti-HA. After identification of HA-positive cells by confocal fluorescence microscopy, cells were processed for SEM. The same cells were identified based on grid positions and analyzed by SEM. (A) Representative SEM images for WSN-infected HA-positive cells are shown. Fluorescence images corresponding to the SEM images are also included. Boxed areas for SEM images are magnified and shown on the right of original images. Alphabetic labels are used to distinguish between the individual boxed areas. (B) The number of ∼100-nm buds identified in SEM images was counted within the same-size area (100 μm 2 in size) in each cell. Data are shown for 10 to 20 cells from three independent experiments. (C) vRNA release efficiency was measured in infected MDM and dTHP1 cell cultures treated with DMSO or Cyto D for 4 h. For panel B, error bars represent standard error of mean. For panel C, error bars represent SD. **, P
    Figure Legend Snippet: Cytochalasin D treatment increases bud formation in MDM to levels comparable to that in dTHP1 cells. dTHP1 cells and MDM were infected with WSN at MOI 0.1. At 14 hpi, cells were treated with vehicle control (DMSO) or 20 μM Cyto D for 4 h before fixation and immunostaining with anti-HA. After identification of HA-positive cells by confocal fluorescence microscopy, cells were processed for SEM. The same cells were identified based on grid positions and analyzed by SEM. (A) Representative SEM images for WSN-infected HA-positive cells are shown. Fluorescence images corresponding to the SEM images are also included. Boxed areas for SEM images are magnified and shown on the right of original images. Alphabetic labels are used to distinguish between the individual boxed areas. (B) The number of ∼100-nm buds identified in SEM images was counted within the same-size area (100 μm 2 in size) in each cell. Data are shown for 10 to 20 cells from three independent experiments. (C) vRNA release efficiency was measured in infected MDM and dTHP1 cell cultures treated with DMSO or Cyto D for 4 h. For panel B, error bars represent standard error of mean. For panel C, error bars represent SD. **, P

    Techniques Used: Infection, Immunostaining, Fluorescence, Microscopy

    37) Product Images from "Assembly and Budding of Ebolavirus"

    Article Title: Assembly and Budding of Ebolavirus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0020099

    VLP Budding Is Dependent on Microtubules 10 μM nocodazole (noc), 1 μM taxol (tax), 10 μg/ml cytochalasin D (cytD), or 5 μM monensin (mon) was added to cells 3 h after they were transfected with plasmids expressing (A) NP, VP24, VP35, and VP40, or (B) VP40 alone. At 16 h post-transfection, proteins in the cell lysates and supernatants were separated by SDS-PAGE and examined by Western blotting with anti-NP and anti-VP40 antibodies. Following nocodazol or taxol treatment, the amounts of both VP40 and NP (A) or VP40 (B) in the supernatants (i.e., efficiency of VLPs budding) were reduced. cont, mock-treated control.
    Figure Legend Snippet: VLP Budding Is Dependent on Microtubules 10 μM nocodazole (noc), 1 μM taxol (tax), 10 μg/ml cytochalasin D (cytD), or 5 μM monensin (mon) was added to cells 3 h after they were transfected with plasmids expressing (A) NP, VP24, VP35, and VP40, or (B) VP40 alone. At 16 h post-transfection, proteins in the cell lysates and supernatants were separated by SDS-PAGE and examined by Western blotting with anti-NP and anti-VP40 antibodies. Following nocodazol or taxol treatment, the amounts of both VP40 and NP (A) or VP40 (B) in the supernatants (i.e., efficiency of VLPs budding) were reduced. cont, mock-treated control.

    Techniques Used: Transfection, Expressing, SDS Page, Western Blot

    38) Product Images from "Cryptococcus neoformans Induces Macrophage Inflammatory Protein 1? (MIP-1?) and MIP-1? in Human Microglia: Role of Specific Antibody and Soluble Capsular Polysaccharide"

    Article Title: Cryptococcus neoformans Induces Macrophage Inflammatory Protein 1? (MIP-1?) and MIP-1? in Human Microglia: Role of Specific Antibody and Soluble Capsular Polysaccharide

    Journal: Infection and Immunity

    doi: 10.1128/IAI.69.3.1808-1815.2001

    Cytochalasin D inhibits internalization of MAb-opsonized C. neoformans but does not alter MIP-1α induction. (A and B) Microglia were incubated with C. neoformans and MAb as described above in the absence (A) or presence (B) of 2 μM cytochalasin D. After a 90-min incubation, cultures were washed to remove nonadherent organisms, suspended in medium with or without cytochalasin D, and allowed to incubate for 16 h. (C) MIP-1α levels in supernatants from these microglial cultures were determined by ELISA as described above.
    Figure Legend Snippet: Cytochalasin D inhibits internalization of MAb-opsonized C. neoformans but does not alter MIP-1α induction. (A and B) Microglia were incubated with C. neoformans and MAb as described above in the absence (A) or presence (B) of 2 μM cytochalasin D. After a 90-min incubation, cultures were washed to remove nonadherent organisms, suspended in medium with or without cytochalasin D, and allowed to incubate for 16 h. (C) MIP-1α levels in supernatants from these microglial cultures were determined by ELISA as described above.

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    39) Product Images from "Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps"

    Article Title: Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.200

    Disruption of NET structure by interfering endogenous polyamine incorporation by monoamines compromises bacterial trapping capacity of the NET. ( a ) Representative images of trapped S. aureus in NETs generated with PMA (I) or in the presence of MDC (II) or BPNH 2 (III). Left panels show NET (red) and FITC-labeled trapped S. aureus (green); right panel shows S. aureus alone. Experiment was repeated three times with neutrophils from independent healthy donors, with similar results. Original magnifications were × 40 and scale bars represent 10 μ m. ( b ) Quantification of the trapping capacity of the NET by bacterial trapping assay. Neutrophils were plated and activated in an ELISA plate for 3 h with PMA (control), PMA and MDC (100 and 250 μ M) or PMA and BPNH 2 (0.5 and 2 mM). Phagocytosis was blocked by cytochalasin D then FITC-labeled S. aureus was given to the cells in 1 : 100 neutrophil:bacterium ratio for additional 1 h. Unbound bacteria were washed away and fluorescence (proportional to the amount of the trapped bacteria) was determined by an ELISA reader. Experiment was repeated three times with neutrophils from independent healthy donors ( n =3) with six technical parallels. Data represent mean±S.D. *** P
    Figure Legend Snippet: Disruption of NET structure by interfering endogenous polyamine incorporation by monoamines compromises bacterial trapping capacity of the NET. ( a ) Representative images of trapped S. aureus in NETs generated with PMA (I) or in the presence of MDC (II) or BPNH 2 (III). Left panels show NET (red) and FITC-labeled trapped S. aureus (green); right panel shows S. aureus alone. Experiment was repeated three times with neutrophils from independent healthy donors, with similar results. Original magnifications were × 40 and scale bars represent 10 μ m. ( b ) Quantification of the trapping capacity of the NET by bacterial trapping assay. Neutrophils were plated and activated in an ELISA plate for 3 h with PMA (control), PMA and MDC (100 and 250 μ M) or PMA and BPNH 2 (0.5 and 2 mM). Phagocytosis was blocked by cytochalasin D then FITC-labeled S. aureus was given to the cells in 1 : 100 neutrophil:bacterium ratio for additional 1 h. Unbound bacteria were washed away and fluorescence (proportional to the amount of the trapped bacteria) was determined by an ELISA reader. Experiment was repeated three times with neutrophils from independent healthy donors ( n =3) with six technical parallels. Data represent mean±S.D. *** P

    Techniques Used: Generated, Labeling, Enzyme-linked Immunosorbent Assay, Fluorescence

    40) Product Images from "Opposite Effects of Insulin on Focal Adhesion Proteins in 3T3-L1 Adipocytes and in Cells Overexpressing the Insulin Receptor"

    Article Title: Opposite Effects of Insulin on Focal Adhesion Proteins in 3T3-L1 Adipocytes and in Cells Overexpressing the Insulin Receptor

    Journal: Molecular Biology of the Cell

    doi:

    Effect of insulin and cytochalasin D on the actin cytoskeleton and tyrosine phosphorylation of FAK and paxillin in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were grown and differentiated on glass coverslips. The cells were serum deprived for 3 h with no other additions (a), with insulin treatment for the last 5 min (b), or with treatment with 2 μM cytochalasin D during the entire 3 h (c). All cells were fixed and detergent permeabilized, and the actin filaments were stained with rhodamine-conjugated phalloidin, as indicated in MATERIALS AND METHODS. (B) 3T3-L1 adipocytes grown in 10-cm dishes were serum deprived for 3 h in the absence (−) or presence (+) of cytochalasin D. Insulin treatments (100 nM) were for the times indicated and were timed so that their total serum deprivation time ended at 3 h. Cell lysates were prepared and immunoprecipitated with anti-PY conjugated to agarose beads (IP, α-PY), as indicated in MATERIALS AND METHODS. Samples were immunoblotted (IB) anti-FAK (α-FAK) and anti-paxillin (α-PAX) as indicated. (C) 3T3-L1 adipocytes were treated with insulin (100 nM) for the times indicated. Cell lysates were prepared and immunoprecipitated with α-FAK or α-PAX antibodies as described in MATERIALS AND METHODS and then immunoblotted with α-PY as indicated. Shown is one experiment representative of two.
    Figure Legend Snippet: Effect of insulin and cytochalasin D on the actin cytoskeleton and tyrosine phosphorylation of FAK and paxillin in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were grown and differentiated on glass coverslips. The cells were serum deprived for 3 h with no other additions (a), with insulin treatment for the last 5 min (b), or with treatment with 2 μM cytochalasin D during the entire 3 h (c). All cells were fixed and detergent permeabilized, and the actin filaments were stained with rhodamine-conjugated phalloidin, as indicated in MATERIALS AND METHODS. (B) 3T3-L1 adipocytes grown in 10-cm dishes were serum deprived for 3 h in the absence (−) or presence (+) of cytochalasin D. Insulin treatments (100 nM) were for the times indicated and were timed so that their total serum deprivation time ended at 3 h. Cell lysates were prepared and immunoprecipitated with anti-PY conjugated to agarose beads (IP, α-PY), as indicated in MATERIALS AND METHODS. Samples were immunoblotted (IB) anti-FAK (α-FAK) and anti-paxillin (α-PAX) as indicated. (C) 3T3-L1 adipocytes were treated with insulin (100 nM) for the times indicated. Cell lysates were prepared and immunoprecipitated with α-FAK or α-PAX antibodies as described in MATERIALS AND METHODS and then immunoblotted with α-PY as indicated. Shown is one experiment representative of two.

    Techniques Used: Staining, Immunoprecipitation

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    Article Snippet: .. Quantification of TNTs Cocultures of CTB‐labeled and unlabeled PC12 cells were treated with different concentrations of cytochalasin D (Sigma–Aldrich) for 24 h and stained with WGA‐AF488 (Invitrogen) for 5 min. Then live‐cell imaging was performed by using a Zeiss Axiovert 200 M fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with a 63 × /1.40 NA oil‐immersion objective and a DAPI/FITC/TRITC filter set. .. For each condition, stacks of 20 images from the bottom to the top of cells were acquired at 400 nm (CTB) and at 488 nm (WGA‐AF488) within 20 min 20 stacks containing at least 350 cells were obtained in each condition.

    Fluorescence:

    Article Title: TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro‐Phagocytic Signals From Apoptotic to Viable Cells
    Article Snippet: .. Quantification of TNTs Cocultures of CTB‐labeled and unlabeled PC12 cells were treated with different concentrations of cytochalasin D (Sigma–Aldrich) for 24 h and stained with WGA‐AF488 (Invitrogen) for 5 min. Then live‐cell imaging was performed by using a Zeiss Axiovert 200 M fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with a 63 × /1.40 NA oil‐immersion objective and a DAPI/FITC/TRITC filter set. .. For each condition, stacks of 20 images from the bottom to the top of cells were acquired at 400 nm (CTB) and at 488 nm (WGA‐AF488) within 20 min 20 stacks containing at least 350 cells were obtained in each condition.

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    Article Title: TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro‐Phagocytic Signals From Apoptotic to Viable Cells
    Article Snippet: .. Quantification of TNTs Cocultures of CTB‐labeled and unlabeled PC12 cells were treated with different concentrations of cytochalasin D (Sigma–Aldrich) for 24 h and stained with WGA‐AF488 (Invitrogen) for 5 min. Then live‐cell imaging was performed by using a Zeiss Axiovert 200 M fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with a 63 × /1.40 NA oil‐immersion objective and a DAPI/FITC/TRITC filter set. .. For each condition, stacks of 20 images from the bottom to the top of cells were acquired at 400 nm (CTB) and at 488 nm (WGA‐AF488) within 20 min 20 stacks containing at least 350 cells were obtained in each condition.

    Microscopy:

    Article Title: TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro‐Phagocytic Signals From Apoptotic to Viable Cells
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    Concentration Assay:

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    other:

    Article Title: Comparative Study of Domoic Acid and Okadaic Acid Induced - Chromosomal Abnormalities in the CACO-2 Cell Line
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    Article Title: Comparative evaluation of the mutagenicity and genotoxicity of smoke condensate derived from Korean cigarettes
    Article Snippet: Dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), 2-aminoanthracene, benzo[a]pyrene and cytochalasin B were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    CtB Assay:

    Article Title: TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro‐Phagocytic Signals From Apoptotic to Viable Cells
    Article Snippet: .. Quantification of TNTs Cocultures of CTB‐labeled and unlabeled PC12 cells were treated with different concentrations of cytochalasin D (Sigma–Aldrich) for 24 h and stained with WGA‐AF488 (Invitrogen) for 5 min. Then live‐cell imaging was performed by using a Zeiss Axiovert 200 M fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with a 63 × /1.40 NA oil‐immersion objective and a DAPI/FITC/TRITC filter set. .. For each condition, stacks of 20 images from the bottom to the top of cells were acquired at 400 nm (CTB) and at 488 nm (WGA‐AF488) within 20 min 20 stacks containing at least 350 cells were obtained in each condition.

    Staining:

    Article Title: TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro‐Phagocytic Signals From Apoptotic to Viable Cells
    Article Snippet: .. Quantification of TNTs Cocultures of CTB‐labeled and unlabeled PC12 cells were treated with different concentrations of cytochalasin D (Sigma–Aldrich) for 24 h and stained with WGA‐AF488 (Invitrogen) for 5 min. Then live‐cell imaging was performed by using a Zeiss Axiovert 200 M fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with a 63 × /1.40 NA oil‐immersion objective and a DAPI/FITC/TRITC filter set. .. For each condition, stacks of 20 images from the bottom to the top of cells were acquired at 400 nm (CTB) and at 488 nm (WGA‐AF488) within 20 min 20 stacks containing at least 350 cells were obtained in each condition.

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    Millipore cytochalasin d
    Elastic modulus of CHO cells treated with nocodazole, trypsin or cytochalasin D. p-values were derived from Microsoft Excel’s Student’s T-test. N=10.
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Elastic modulus of CHO cells treated with nocodazole, trypsin or cytochalasin D. p-values were derived from Microsoft Excel’s Student’s T-test. N=10.

    Journal: Methods (San Diego, Calif.)

    Article Title: Rearrangement of microtubule network under biochemical and mechanical stimulations

    doi: 10.1016/j.ymeth.2013.02.014

    Figure Lengend Snippet: Elastic modulus of CHO cells treated with nocodazole, trypsin or cytochalasin D. p-values were derived from Microsoft Excel’s Student’s T-test. N=10.

    Article Snippet: Nocodazole (Calbiochem) and cytochalasin D (Calbiochem) were added to the cell medium to achieve final concentrations of 20 μM and 5 μM, respectively.

    Techniques: Derivative Assay

    Activation of the integrin signal transduction pathway is crucial for EBV entry into memory B cells. (A and B) Inhibition of signaling molecules c-Src, PI3K, and FAK, activated downstream of integrin, impacts the susceptibility of tonsillar memory B cells to EBV infection by B95.8EBfaV-GFP viral supernatants. Inhibitors used were AG-82 (FAK), PP2 (c-Src), wortmannin (PI3K), and Ly-294002 (PI3K). Cytochalasin D was included to show the dependence of infection susceptibility of memory B cells on actin cytoskeleton depolymerization. Cells were counterstained for CD19 and CD27 to identify memory B cells and determine their infection frequency by flow cytometry. Results shown are from one representative experiment of two experiments.

    Journal: Journal of Virology

    Article Title: ?1 Integrin Expression Increases Susceptibility of Memory B Cells to Epstein-Barr Virus Infection ▿

    doi: 10.1128/JVI.02675-09

    Figure Lengend Snippet: Activation of the integrin signal transduction pathway is crucial for EBV entry into memory B cells. (A and B) Inhibition of signaling molecules c-Src, PI3K, and FAK, activated downstream of integrin, impacts the susceptibility of tonsillar memory B cells to EBV infection by B95.8EBfaV-GFP viral supernatants. Inhibitors used were AG-82 (FAK), PP2 (c-Src), wortmannin (PI3K), and Ly-294002 (PI3K). Cytochalasin D was included to show the dependence of infection susceptibility of memory B cells on actin cytoskeleton depolymerization. Cells were counterstained for CD19 and CD27 to identify memory B cells and determine their infection frequency by flow cytometry. Results shown are from one representative experiment of two experiments.

    Article Snippet: The inhibitors PP2, wortmannin, cytochalasin D, and AG-82 were from Calbiochem (Dietikon, Switzerland), and Ly-294002 was from Sigma-Aldrich.

    Techniques: Activation Assay, Transduction, Inhibition, Infection, Flow Cytometry, Cytometry

    Mechanisms of amphotericin B-, nystatin-, and natamycin-induced NLRP3 activation. (A) IL-1β ELISA for wildtype and ASC-deficient BMDCs stimulated with the antimycotic drugs as indicated. Note that IL-1β secretion was dependent on the presence of NLRP3 and ASC for all drugs. (B) LPS prestimulated wildtype BMDCs were stimulated with the drugs in the presence or absence of the caspase inhibitor Z-VAD-FMK; supernatants were collected 6 hours later, and IL-1β release was assessed by ELISA. (C) LPS-primed wild type BMDCs were stimulated with antifungals in serum free buffer with or without 75 mM KCl or NaCl. IL-1β secretion was measured in supernatants after 6 hours (D). Wildtype or P2X7-deficient BMDCs were primed with LPS and exposed 50 µg/ml of each Amphotericin B, nystatin, and natamycin. IL-1β secretion was measured in supernatants after 6 hours of stimulation. ATP was used as control. (E) and (F) LPS-prestimulated wild type BMDCs were treated with cytochalasin D, NAC and the cathepsin inhibitor CA-07-Me for 30 min followed by stimulation with the antimycotic drugs for 6 hours. IL-1β secretion was measured in supernatants by ELISA. Data are means ± SD from three independent experiments all performed in triplicate. p

    Journal: PLoS ONE

    Article Title: Polyene Macrolide Antifungal Drugs Trigger Interleukin-1? Secretion by Activating the NLRP3 Inflammasome

    doi: 10.1371/journal.pone.0019588

    Figure Lengend Snippet: Mechanisms of amphotericin B-, nystatin-, and natamycin-induced NLRP3 activation. (A) IL-1β ELISA for wildtype and ASC-deficient BMDCs stimulated with the antimycotic drugs as indicated. Note that IL-1β secretion was dependent on the presence of NLRP3 and ASC for all drugs. (B) LPS prestimulated wildtype BMDCs were stimulated with the drugs in the presence or absence of the caspase inhibitor Z-VAD-FMK; supernatants were collected 6 hours later, and IL-1β release was assessed by ELISA. (C) LPS-primed wild type BMDCs were stimulated with antifungals in serum free buffer with or without 75 mM KCl or NaCl. IL-1β secretion was measured in supernatants after 6 hours (D). Wildtype or P2X7-deficient BMDCs were primed with LPS and exposed 50 µg/ml of each Amphotericin B, nystatin, and natamycin. IL-1β secretion was measured in supernatants after 6 hours of stimulation. ATP was used as control. (E) and (F) LPS-prestimulated wild type BMDCs were treated with cytochalasin D, NAC and the cathepsin inhibitor CA-07-Me for 30 min followed by stimulation with the antimycotic drugs for 6 hours. IL-1β secretion was measured in supernatants by ELISA. Data are means ± SD from three independent experiments all performed in triplicate. p

    Article Snippet: Inhibitors such as Z-VAD-FMK (20 µM, all from Invivogen), cytochalasin D (5 µM), CA-074-Me (10 µM, Calbiochem, Darmstadt, Germany), NAC (10mM, Sigma) and Quinidine (250 µM, Sigma) were added 30min before antifungals stimulation.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

    Examples ( A–C ) and summary data ( D–F ) showing the effects of the actin polymerization inhibitors cytochalasin-D (Cyto-D; 0.2 μM; A and D ) and latrunculin-B (Latr-B; 2 μM; B and E ), and the RhoA kinase inhibitor H-1152 (0.3

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Active tension adaptation at a shortened arterial muscle length: inhibition by cytochalasin-D

    doi: 10.1152/ajpheart.00009.2010

    Figure Lengend Snippet: Examples ( A–C ) and summary data ( D–F ) showing the effects of the actin polymerization inhibitors cytochalasin-D (Cyto-D; 0.2 μM; A and D ) and latrunculin-B (Latr-B; 2 μM; B and E ), and the RhoA kinase inhibitor H-1152 (0.3

    Article Snippet: Latrunculin-B, cytochalasin-D, and H-1152 were from Calbiochem (La Jolla, CA).

    Techniques: