cytochalasin d Millipore Search Results


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  • 99
    Millipore cytochalasin d
    Gliding Motility and Traversal Capacity of Wt and Δ p52  sporozoites. (A) Representative immunofluorescence staining with anti-PfCSP antibodies of the trails produced by Wt and mutant sporozoites deficient in P52 expression (Δ p52-1 and  Δ p52-2 ) as well as Wt sporozoites, treated with cytochalasin D, an inhibitor of sporozoite motility. Characteristic circles of gliding motility are present in Wt and mutant lines, and absent in Wt sporozoites that have been treated with cytochalasin D. (B) Gliding motility of  P. falciparum  Wt (cytochalsin D treated and untreated) and mutant sporozoites as assessed by the capacity to produce the characteristic circles (see A). (C) Cell traversal ability of  P. falciparum  Wt and mutant sporozoites as determined by FACS counting of Dextran positive hepG2 cells. Dex: hepatocytes cultured in the presence of Dextran but without the addition of sporozoites.
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9500 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nocodazole
    Eukaryotic cytoskeleton is important for K. pneumoniae invasion. Invasion assays of K. pneumoniae Ca0438 using Caco-2 cells were performed in the presence of compounds that interfered with host actin (cytochalasin D) or microtubules <t>(nocodazole).</t> Mock invasion in the absence of inhibitors was defined as 100% relative invasiveness. Data are presented as means ± SEM. *, P
    Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15733 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore latrunculin a
    RSV infection induces actin rearrangement. (A). RSV (moi ∼0.5) was bound to HeLa cells at 4°C, unbound virus was removed and cells warmed to 37°C, fixed at indicated times, and stained with phalloidin-AF488 (pseudocolored white) and anti-F-AF647 (red) antibody. Images represent Z-stack projections acquired with a confocal microscope. Arrowheads show actin blebs formed at the cell surface. (B). RSV (moi ∼30) was incubated with HeLa cells for 30 or 120 min at 37°C. Samples were processed according to the kit manufacturer's protocol (Cytoskeleton Inc.). Controls included mock-treated cells and cells either treated with F actin enhancer or F actin depolymerizing agent. (left) The F and G actin fractions were resolved by SDS-PAGE and western blots probed with anti-actin antibody. (right) Quantification of actin protein bands intensities by densitometry. (C–F). HeLa cells were pretreated with solvent (MOCK), cytochalasin D (CytoD), <t>latrunculin</t> A (LatA), jasplakinolide (Jas), nocodazole (Noc), taxol (Tax), NCS23766, pirl1, IPA-3, wiskostatin (Wisko), CK-869, CT04, Y24632 at indicated concentrations and each inhibitor was continuously present during following steps of the experiment: (C, E). Cells where infected with RSV (moi ∼3) or SFV-ZsGreen (moi ∼0.5) for up to 6 hours before FACS analysis of GFP expressing cells. (D, F). RSV (moi ∼3) was bound to the cells at 4°C followed by 1 h of internalization at 37°C. Cells were trypsinized, fixed and stained with anti-N-AF488 antibody, and the MFI of AF-488 measured by FACS.
    Latrunculin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2481 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cytarabine
    The effect of <t>cytarabine</t> alone compared with sorafenib plus cytarabine on leukemic cell colonization in the spleen and on spleen size. NOD-SCID-IL2R γ null mice were injected with luciferase-labeled U937 cells and treated 3 days later with cytarabine
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    Millipore blebbistatin
    P2X 7 R mediates LL-37 internalization by human macrophages. ( A and B ) dTHP-1 cells were pretreated with P2X 7 R inhibitors KN-62 (1 μM) or oxATP (100 μM) for 1 h and incubated with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). (A) A representative FACS plot. ( C and D ) Control (transfected with nontarget shRNA) and P2X 7 R-KD (transfected with human P2X 7 R-targeted shRNA) dTHP-1 cells were incubated with 10 μg/ml of FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was analyzed by flow cytometry ( n = 5). (C) One representative FACS plot. ( E ) WT, control, and P2X 7 R-KD THP-1 cells were pretreated with P2X 7 R inhibitor KN-62 (1 μM) for 1 h before incubation with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( F ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and P2X 7 R was visualized using a confocal microscope. ( G ) Control and P2X 7 R-KD dTHP-1 cells were pretreated with nystatin (10 μg/ml) or dynasore (20 μM), respectively, at 37°C for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 5). ( H ) dTHP-1 cells were pretreated with BzATP (100 μM, specific agonist of P2X 7 R), <t>(-)-blebbistatin</t> (200 μM, inhibitor of nonmuscle myosin II), 10 PanX (1 μM, peptide antagonist of Panx-1), FIPI (1 μM, inhibitor of PLD isoforms), wortmannin (1 μM, specific inhibitor of PI 3 K), geldanamycin (1 μM, inhibitor of hsp90), U0126 (1 μM, inhibitor of ERK MAPK), or SB203580 (1 μM, inhibitor of p38 MAPK), followed by incubation with FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( I ) The colocalization of LL-37/P2X 7 R complex and caveolin-1 or clathrin was visualized using a confocal microscope. Images in (I a ) and (I b ) are enlargements of the boxed areas. A total of 10 μg/ml of LL-37 was used in all treatments, and the confocal images are representative of at least three experiments. Scale bars, 10 μm. * p
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    Millipore propidium iodide
    Knockdown of TNFAIP8 induces epithelial ovarian cancer cell cycle arrest. (A) Following siNC or siTNFAIP8 transfection for 48 h, A2780s or A2780cp cells were collected for <t>propidium</t> iodide staining, followed by flow cytometry analysis. The percentage of cells in each stage was analyzed. *P
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    Millipore triton x 100
    Localization of F-actin in MCF-7/MR cells and inhibition of actin polymerization by CytD. (A) Cells grown for 7 days on glass cover slips were fixed, permeabilized with <t>Triton-X-100</t> and reacted with anti-ZO-1 antibody (a and d) and with rhodamine phalloidin (b and e) to follow F-actin. The localization of actin and ZO-1 at the EVs surface is shown for EVs formed between two (a–c) and/or multiple attached cells (d–f). Arrows denote the localization of cell-cell attachment zones (belt-like structures). (B) Cells were either untreated (a–c) or treated with CytD (10 µg/ml) for 30 min at 37°C (d–f), washed and reacted as in panel A, to visualize ZO-1 (a and d) and F-actin (b and e). (C) Cells were treated as described in panel B and then stained for ABCG2 (a and d) and F-actin (b and e). (D) Cells were grown in riboflavin (B2)-deficient medium for 48 hr prior to CytD treatment to avoid riboflavin accumulation in EVs. Cells were then washed and transferred to riboflavin-containing medium for an additional 24 hr to examine the riboflavin accumulation capacity (a–c). Untreated cells grown continuously in medium containing (d–f) or lacking riboflavin (g–i) served as controls. Arrows denote the location of EVs. Cells were analyzed using a Zeiss inverted Cell-Observer microscope at a magnification of ×630 (A–C) or ×200 (D). The merged images including DAPI staining (panels A–C), were generated using Cell-Observer software.
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    Millipore wortmannin
    PI3K inhibition reduced morphine-induced microglial migration. Primary microglial cells were treated with two PI3K inhibitors, <t>wortmannin</t> and LY294002, for 1 h then treated for 2 h with morphine and allowed to migrate toward 10 μ m ADP. Migrated
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    Millipore jasplakinolide
    Intracellular signaling mediates adhesion to anti-TCR treated plates. (A) Jurkat E6.1 cells were stimulated with various concentrations of the immobilized anti-TCR antibodies OKT3 and IP26 for 30 minutes. Total changes in TCR-inducible tyrosine phosphorylation were detected by immunoblot (left panel). TCR-induced adhesion in response to these antibodies or two non-specific antibodies (IgG2a and total mouse IgG) was also quantified and is graphed as the average three experiments ± SD (right panel). These data are shown as the average of three experiments (B) Jurkat E6.1 cells (left panel) and hAPBTs (right panel) were stained and pretreated with DMSO or the actin cytoskeleton inhibitors Cytochalasin D (Cyto D) and Latrunculin B (Lat B) for 30 minutes or <t>jasplakinolide</t> (Jasp) for 15 minutes at 37°C. The cells were applied to an anti-TCR coated RIA/EIA plate and allowed to adhere for 30 minutes at 37°C. The data were normalized such that the DMSO samples were set to 100%. The normalized results from three independent experiments were then graphed. ** p≤0.01 *** p≤0.001.
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    Millipore porcine stomach
    Intracellular signaling mediates adhesion to anti-TCR treated plates. (A) Jurkat E6.1 cells were stimulated with various concentrations of the immobilized anti-TCR antibodies OKT3 and IP26 for 30 minutes. Total changes in TCR-inducible tyrosine phosphorylation were detected by immunoblot (left panel). TCR-induced adhesion in response to these antibodies or two non-specific antibodies (IgG2a and total mouse IgG) was also quantified and is graphed as the average three experiments ± SD (right panel). These data are shown as the average of three experiments (B) Jurkat E6.1 cells (left panel) and hAPBTs (right panel) were stained and pretreated with DMSO or the actin cytoskeleton inhibitors Cytochalasin D (Cyto D) and Latrunculin B (Lat B) for 30 minutes or <t>jasplakinolide</t> (Jasp) for 15 minutes at 37°C. The cells were applied to an anti-TCR coated RIA/EIA plate and allowed to adhere for 30 minutes at 37°C. The data were normalized such that the DMSO samples were set to 100%. The normalized results from three independent experiments were then graphed. ** p≤0.01 *** p≤0.001.
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    Millipore paclitaxel
    Effect of <t>Paclitaxel</t> ( Pac ) on cell deformation. HL‐60 cells were incubated with varying concentrations of Pac and cell deformation was observed using RT‐DC. (a) Scatter plots of HL‐60 cell populations incubated without (Control) and with 1 μM Pac . The contour lines compare the 50% event density of the control (black) vs Pac (green). The inset shows the reservoir measurement for reference. (b) Representative fluorescence images (microtubule, green and DNA, blue). Scale bar is 10 μm. (c) Dose–response graph showing mean RD values of three replicates of HL‐60 cell population treated with increasing concentrations of Pac that allowed for the extraction of a half‐maximal concentration (EC 50 ) for the effect of Pac on the deformation of HL‐60 cells. Shaded red area indicates 95% confidence interval of the fit. Tangential slope at the inflection point of the sigmoidal fit function is 0.3 with Hill Coefficient 1.61. Error bars are standard error of the mean (SEM). All experiments were carried out at a flow rate of 0.04 μL·s −1 in a 20 × 20 × 300 μm (w × h × l) channel. p values denote *** p ≤ 0.001 and ns, not significant. [Color figure can be viewed at wileyonlinelibrary.com ]
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    Millipore pbs
    Effect of <t>Paclitaxel</t> ( Pac ) on cell deformation. HL‐60 cells were incubated with varying concentrations of Pac and cell deformation was observed using RT‐DC. (a) Scatter plots of HL‐60 cell populations incubated without (Control) and with 1 μM Pac . The contour lines compare the 50% event density of the control (black) vs Pac (green). The inset shows the reservoir measurement for reference. (b) Representative fluorescence images (microtubule, green and DNA, blue). Scale bar is 10 μm. (c) Dose–response graph showing mean RD values of three replicates of HL‐60 cell population treated with increasing concentrations of Pac that allowed for the extraction of a half‐maximal concentration (EC 50 ) for the effect of Pac on the deformation of HL‐60 cells. Shaded red area indicates 95% confidence interval of the fit. Tangential slope at the inflection point of the sigmoidal fit function is 0.3 with Hill Coefficient 1.61. Error bars are standard error of the mean (SEM). All experiments were carried out at a flow rate of 0.04 μL·s −1 in a 20 × 20 × 300 μm (w × h × l) channel. p values denote *** p ≤ 0.001 and ns, not significant. [Color figure can be viewed at wileyonlinelibrary.com ]
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    Millipore bafilomycin a1
    Entry of RVFV ns depends on vacuolar acidification. (A) BHK-21 or A549 cells were pretreated for 1 h with different concentrations of <t>bafilomycin</t> A1 or ammonium chloride (NH 4 Cl) and subsequently infected with RVFV ns (MOI of ∼0.1) or VSV ns (MOI
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    Millipore bsa
    Representative chromatograms and UV–vis absorption spectra showing the complexation of metal ions and reduction of cytochrome c. Panel (A) shows the complexation of Pb by <t>sEPS1</t> (alginate and <t>BSA)</t> on the anode (orange), the complexation of Fe by sEPS1 on the cathode (blue), and the complexation of Pb when Cyto. c (alginate, BSA, and cytochrome c) was on the anode (red). Panel (B) shows the contrast of UV–vis absorption spectra from cytochrome c before and after corrosion tests on the anode and cathode in which anaerobic or aerobic states the condition on the cathode side and those after the colon indicate the location of Cyto. c.
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    Millipore dapi
    Numbers of circulating endothelial cells (defined as CD45 NEG , CD146 POS , <t>CD133</t> NEG and <t>DAPI</t> bright with viable nuclei), with Day 1 draw immediately prior to initiation of first cycle of treatment and the final draw at time of disease progression.
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    Millipore flag peptide
    Neutralizing effect of mAb 286, mapping of its binding site, and analysis of binding to <t>VEGF-D</t> variants with mutated residues in N-terminal α-helix. A , the capacity of mAb 286 to block binding and cross-linking, by VEGF-DΔNΔC, of chimeric receptors containing VEGFR-2 ( left ) or VEGFR-3 ( right ) extracellular domains was assessed in bioassays (see “Experimental Procedures”). Also included were neutralizing mAb VD1, which binds loop 2 of VEGF-DΔNΔC, and mAb VD4, which binds, but does not neutralize, VEGF-DΔNΔC ( 39 ). B , peptide-based mapping of the mAb 286 binding site in VEGF-DΔNΔC by ELISA (see “Experimental Procedures”). The ratio of signal to background for the interaction of mAb 286 with immobilized peptides is shown on the y axis of the graph, and the x axis indicates the identifier numbers of peptides. Top box above the graph , amino acid sequence for the VEGF homology domain of human VEGF-D; N-terminal residue (phenylalanine) is number 89, and the C-terminal residue (arginine) is 205. Bottom box above the graph , examples of peptides used in mapping (mAb 286 binding site is in a rectangle ). The <t>FLAG</t> sequence is shown in boldface type in peptide 36, which lacks the VEGF-D-derived sequence, and was the negative control. C , detection of VEGF-DΔNΔC variants by Western blotting under reducing and denaturing conditions using mAb 286 ( top ) or M2 anti-FLAG mAb as a positive control ( bottom ). Each well contained 30 ng of purified protein. VEGF-D , VEGF-DΔNΔC; variants of this protein each have one residue mutated to alanine, as indicated. Positions of molecular mass markers (in kDa) are shown to the left . The histogram under the blots shows intensities of bands for VEGF-D variants (mean ± S.D.) relative to the intensity of the band for VEGF-DΔNΔC, as determined from Western blots with mAb 286. D , analysis of mAb 286 binding to VEGF-DΔNΔC variants by ELISA. M2 was used for capture and mAb 286 for detection; the y axis shows binding of variant proteins compared with VEGF-DΔNΔC (the latter defined as 100% binding), and the x axis lists VEGF-D variants. Equal amounts of VEGF-DΔNΔC and variants were used. For A , B , and D , assays were conducted three times. Columns , mean; error bars , S.D.
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    Millipore colchicine
    Neutralizing effect of mAb 286, mapping of its binding site, and analysis of binding to <t>VEGF-D</t> variants with mutated residues in N-terminal α-helix. A , the capacity of mAb 286 to block binding and cross-linking, by VEGF-DΔNΔC, of chimeric receptors containing VEGFR-2 ( left ) or VEGFR-3 ( right ) extracellular domains was assessed in bioassays (see “Experimental Procedures”). Also included were neutralizing mAb VD1, which binds loop 2 of VEGF-DΔNΔC, and mAb VD4, which binds, but does not neutralize, VEGF-DΔNΔC ( 39 ). B , peptide-based mapping of the mAb 286 binding site in VEGF-DΔNΔC by ELISA (see “Experimental Procedures”). The ratio of signal to background for the interaction of mAb 286 with immobilized peptides is shown on the y axis of the graph, and the x axis indicates the identifier numbers of peptides. Top box above the graph , amino acid sequence for the VEGF homology domain of human VEGF-D; N-terminal residue (phenylalanine) is number 89, and the C-terminal residue (arginine) is 205. Bottom box above the graph , examples of peptides used in mapping (mAb 286 binding site is in a rectangle ). The <t>FLAG</t> sequence is shown in boldface type in peptide 36, which lacks the VEGF-D-derived sequence, and was the negative control. C , detection of VEGF-DΔNΔC variants by Western blotting under reducing and denaturing conditions using mAb 286 ( top ) or M2 anti-FLAG mAb as a positive control ( bottom ). Each well contained 30 ng of purified protein. VEGF-D , VEGF-DΔNΔC; variants of this protein each have one residue mutated to alanine, as indicated. Positions of molecular mass markers (in kDa) are shown to the left . The histogram under the blots shows intensities of bands for VEGF-D variants (mean ± S.D.) relative to the intensity of the band for VEGF-DΔNΔC, as determined from Western blots with mAb 286. D , analysis of mAb 286 binding to VEGF-DΔNΔC variants by ELISA. M2 was used for capture and mAb 286 for detection; the y axis shows binding of variant proteins compared with VEGF-DΔNΔC (the latter defined as 100% binding), and the x axis lists VEGF-D variants. Equal amounts of VEGF-DΔNΔC and variants were used. For A , B , and D , assays were conducted three times. Columns , mean; error bars , S.D.
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    Millipore glutathione gsh
    HDACi promote DNA damage by DSBs formation in glioma cell lines. ( a ) Analysis of DNA DSB formation by immunofluorescence of γH2AX-positive nuclei in glioma cells treated for 48 h with 10 mM VPA, 10 μ M SAHA or 100 μ M <t>TMZ.</t> Data are mean±S.E.M. from five independent experiments. ( b ) WST-1 cell viability assay on glioma cell lines exposed to increasing concentrations of TMZ (from 10 to 500 μ M) for 48 h. Data shown are mean±S.E.M. from three independent experiments. ( c ) Analysis of γH2AX-positive nuclei on glioma cells treated with 5 μ M Q-VD-OPh, 10 μ M SAHA or both combined for 48 h. Bars depict mean±S.E.M. from four independent experiments. ( d ) ROS production measured by flow cytometry on glioma cells treated with 10 mM VPA, 10 μ M SAHA or with 10 μ M SAHA in the presence of 10 mM <t>GSH.</t> Data are mean±S.E.M. from four independent experiments. ( e ) Analysis of γH2AX-positive nuclei on glioma cells treated with 10 μ M SAHA or 100 μ M TMZ in the presence or absence of 15 mM N -acetyl- L -cysteine (NAC). Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed by the Student's T -Test, * P
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    Millipore chlorpromazine
    HDACi promote DNA damage by DSBs formation in glioma cell lines. ( a ) Analysis of DNA DSB formation by immunofluorescence of γH2AX-positive nuclei in glioma cells treated for 48 h with 10 mM VPA, 10 μ M SAHA or 100 μ M <t>TMZ.</t> Data are mean±S.E.M. from five independent experiments. ( b ) WST-1 cell viability assay on glioma cell lines exposed to increasing concentrations of TMZ (from 10 to 500 μ M) for 48 h. Data shown are mean±S.E.M. from three independent experiments. ( c ) Analysis of γH2AX-positive nuclei on glioma cells treated with 5 μ M Q-VD-OPh, 10 μ M SAHA or both combined for 48 h. Bars depict mean±S.E.M. from four independent experiments. ( d ) ROS production measured by flow cytometry on glioma cells treated with 10 mM VPA, 10 μ M SAHA or with 10 μ M SAHA in the presence of 10 mM <t>GSH.</t> Data are mean±S.E.M. from four independent experiments. ( e ) Analysis of γH2AX-positive nuclei on glioma cells treated with 10 μ M SAHA or 100 μ M TMZ in the presence or absence of 15 mM N -acetyl- L -cysteine (NAC). Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed by the Student's T -Test, * P
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    Millipore cytosine β d arabinofuranoside
    Timeline (from top to bottom) for neuronal differentiation of mouse embryonic stem cells (ES cells) into an expandable population of cerebellar neurons. Graphs show the stem cell stage (top panel: ES stage), the induction stage of embryoid bodies (EB) in SFEBq culture (EB differentiation stage), an intermediate expansion or proliferation stage of neural progenitor cells (NPCs) through stable passaging on laminin in NPCs medium (NPC expansion stage), and finally the maturation in vitro in NS21 medium ( in vitro maturation stage) and maturation in adult wild type mouse cerebella (bottom panel: in vivo maturation stage). P2 indicates passage 2 (of expansion stage); Arac indicates cytosine <t>β-D-arabinofuranoside;</t> Scale bars indicate 100 μm.
    Cytosine β D Arabinofuranoside, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1339 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore brefeldin a
    Infection with live tachyzoites is not required for TLR11-mediated IL-12 production from DC. A , sort-purified CD11c + splenic DCs were incubated with YFP-expressing tachyzoites alone or in the presence of the indicated concentrations of cytochalasin D for 20 h. The percentage of infected cells (CD11c + YFP + ) was assayed by flow cytometry gated on DCs. B , amount of IL-12/23p40 present in supernatants of DCs co-cultured with tachyzoites (as described in A ) was measured by ELISA. The data shown are representative of six experiments. C , CD11c + splenic DCs were incubated with YFP-expressing tachyzoites (2:1 ratio) for 20 h, and <t>brefeldin</t> A was added during the last 6 h of the culture. IL-12 production was analyzed by intracellular cytokine staining. As a positive control, DCs were stimulated with recombinant T. gondii profilin (1 μg/ml) as described above. D , mice were infected intraperitoneally with 10 5 YFP-expressing tachyzoites. Twenty hours later spleen was isolated and the percentage of IL-12 producing and infected cells (YFP + ) was analyzed by intracellular cytokine staining after incubation of splenocytes for 6 h in the presence of brefeldin A. E , splenocytes depleted of DCs ( Sp ) were placed in the bottom of a Transwell plate (separating membrane: 0.4-μm pores) and stimulated with live parental profilin-sufficient strain tachyzoites ( Toxo , ΔTgPRFe/TgPRFi strain), recombinant profilin (1 μg/ml), or profilin-deficient strain tachyzoites ( TgPRF KO Toxo , ΔTgPRFe/TgPRFi strain+ATc) in the presence or absence of DCs (upper portion of Transwell plate). As a positive control, DCs were cultivated with tachyzoites or recombinant profilin without a separating membrane. IL-12/23p40 present in supernatants was measured by ELISA. The data shown are representative of three performed experiments. Error bars , S.D.
    Brefeldin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33643 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore methyl β cyclodextrin
    Effect of endosomal inhibitors on the entry of  Mr Nvc VLPs into Sf9 cells. Sf9 cells were pre-incubated with different endosomal inhibitors: (A(iii)) cytochalasin D (2 µM), (A(iv)) NH 4 Cl (10 mM), (A(v)) CPZ (50 µM), (A(vi)) methyl- β -cyclodextrin (2 mM) and (A(vii)) genistein (100 µM).  Mr Nvc VLPs labelled with NHS-fluorescein (F- Mr Nvc VLPs; 25 mg/ml) were added to each pre-treated sample and incubated for 16 h in the presence of endosomal inhibitors. Bars, 20 µm. (A(ii)) Sf9 cells added with F- Mr Nvc VLPs but without any inhibitor serve as positive control, whereas (A(i)) Sf9 cells without any inhibitor nor F- Mr Nvc VLPs serve as negative control. (B) MTT assay showing the viability of Sf9 cells in the presence of inhibitors.
    Methyl β Cyclodextrin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0003549

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

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

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

    Monocyte and endothelial cell binding of MN10021 and MN20054. A. Human blood monocytes, isolated as described in Methods, were adjusted to 5×10 6 /ml in PBS (0.5% BSA, pH 7.5). Two-tenths ml of cell suspension was added to each of triplicate 12×75 mm polypropylene tubes and the tubes were incubated for 60 min at 37°C with 20 pmol of [ 125 I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, a 100-fold molar excess of MN10021, or a 100-fold molar excess of MN20050. The contents of each tube were then layered onto 0.8 ml of 10% sucrose in Eppendorf tubes, centrifuged, and the cell-associated radioactivity determined by gamma scintillation spectrophotometry of the cell pellet. B. Binding/uptake of [ 125 I]-MN20054 by human monocytes was performed as described for panel A with the following exceptions: (1) half the cells were pretreated for 30 min at 37°C with cytochalasin B to inhibit endocytosis and (2) competition was performed only with a 100-fold molar excess of MN10021. C. HUVEC were grown to confluence in 12-well tissue culture plates. To each of triplicate wells was added 20 pmol of [ 125 I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, a 100-fold molar excess of MN10021, or a 100-fold molar excess of MN20050. The plates were incubated for 60 min at 37°C with gentle rocking, the wells aspirated and quickly washed 3X with 2.0 ml each of ice-cold PBS/BSA and 1.0 ml of 1.0 N NaOH added to each well to solubilize the cells. Nine-tenths ml of solubilized cells was then removed from each well and cell-associated radioactivity determined by gamma scintillation spectrophotometry.

    Journal: PLoS ONE

    Article Title: Anti-Inflammatory and Vasoprotective Activity of a Retroviral-Derived Peptide, Homologous to Human Endogenous Retroviruses: Endothelial Cell Effects

    doi: 10.1371/journal.pone.0052693

    Figure Lengend Snippet: Monocyte and endothelial cell binding of MN10021 and MN20054. A. Human blood monocytes, isolated as described in Methods, were adjusted to 5×10 6 /ml in PBS (0.5% BSA, pH 7.5). Two-tenths ml of cell suspension was added to each of triplicate 12×75 mm polypropylene tubes and the tubes were incubated for 60 min at 37°C with 20 pmol of [ 125 I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, a 100-fold molar excess of MN10021, or a 100-fold molar excess of MN20050. The contents of each tube were then layered onto 0.8 ml of 10% sucrose in Eppendorf tubes, centrifuged, and the cell-associated radioactivity determined by gamma scintillation spectrophotometry of the cell pellet. B. Binding/uptake of [ 125 I]-MN20054 by human monocytes was performed as described for panel A with the following exceptions: (1) half the cells were pretreated for 30 min at 37°C with cytochalasin B to inhibit endocytosis and (2) competition was performed only with a 100-fold molar excess of MN10021. C. HUVEC were grown to confluence in 12-well tissue culture plates. To each of triplicate wells was added 20 pmol of [ 125 I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, a 100-fold molar excess of MN10021, or a 100-fold molar excess of MN20050. The plates were incubated for 60 min at 37°C with gentle rocking, the wells aspirated and quickly washed 3X with 2.0 ml each of ice-cold PBS/BSA and 1.0 ml of 1.0 N NaOH added to each well to solubilize the cells. Nine-tenths ml of solubilized cells was then removed from each well and cell-associated radioactivity determined by gamma scintillation spectrophotometry.

    Article Snippet: In studies to determine how much of the uptake was due to endocytosis, binding/uptake was performed as described above with the exception that half of the monocytes were pretreated for 30 min at 37°C with cytochalasin B (10 µg/ml; Sigma; St. Louis, MO).

    Techniques: Binding Assay, Isolation, Incubation, Radioactivity, Spectrophotometry

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

    Journal: Infection and Immunity

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

    doi: 10.1128/IAI.02345-14

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

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

    Techniques:

    RSV infection induces actin rearrangement. (A). RSV (moi ∼0.5) was bound to HeLa cells at 4°C, unbound virus was removed and cells warmed to 37°C, fixed at indicated times, and stained with phalloidin-AF488 (pseudocolored white) and anti-F-AF647 (red) antibody. Images represent Z-stack projections acquired with a confocal microscope. Arrowheads show actin blebs formed at the cell surface. (B). RSV (moi ∼30) was incubated with HeLa cells for 30 or 120 min at 37°C. Samples were processed according to the kit manufacturer's protocol (Cytoskeleton Inc.). Controls included mock-treated cells and cells either treated with F actin enhancer or F actin depolymerizing agent. (left) The F and G actin fractions were resolved by SDS-PAGE and western blots probed with anti-actin antibody. (right) Quantification of actin protein bands intensities by densitometry. (C–F). HeLa cells were pretreated with solvent (MOCK), cytochalasin D (CytoD), latrunculin A (LatA), jasplakinolide (Jas), nocodazole (Noc), taxol (Tax), NCS23766, pirl1, IPA-3, wiskostatin (Wisko), CK-869, CT04, Y24632 at indicated concentrations and each inhibitor was continuously present during following steps of the experiment: (C, E). Cells where infected with RSV (moi ∼3) or SFV-ZsGreen (moi ∼0.5) for up to 6 hours before FACS analysis of GFP expressing cells. (D, F). RSV (moi ∼3) was bound to the cells at 4°C followed by 1 h of internalization at 37°C. Cells were trypsinized, fixed and stained with anti-N-AF488 antibody, and the MFI of AF-488 measured by FACS.

    Journal: PLoS Pathogens

    Article Title: Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein

    doi: 10.1371/journal.ppat.1003309

    Figure Lengend Snippet: RSV infection induces actin rearrangement. (A). RSV (moi ∼0.5) was bound to HeLa cells at 4°C, unbound virus was removed and cells warmed to 37°C, fixed at indicated times, and stained with phalloidin-AF488 (pseudocolored white) and anti-F-AF647 (red) antibody. Images represent Z-stack projections acquired with a confocal microscope. Arrowheads show actin blebs formed at the cell surface. (B). RSV (moi ∼30) was incubated with HeLa cells for 30 or 120 min at 37°C. Samples were processed according to the kit manufacturer's protocol (Cytoskeleton Inc.). Controls included mock-treated cells and cells either treated with F actin enhancer or F actin depolymerizing agent. (left) The F and G actin fractions were resolved by SDS-PAGE and western blots probed with anti-actin antibody. (right) Quantification of actin protein bands intensities by densitometry. (C–F). HeLa cells were pretreated with solvent (MOCK), cytochalasin D (CytoD), latrunculin A (LatA), jasplakinolide (Jas), nocodazole (Noc), taxol (Tax), NCS23766, pirl1, IPA-3, wiskostatin (Wisko), CK-869, CT04, Y24632 at indicated concentrations and each inhibitor was continuously present during following steps of the experiment: (C, E). Cells where infected with RSV (moi ∼3) or SFV-ZsGreen (moi ∼0.5) for up to 6 hours before FACS analysis of GFP expressing cells. (D, F). RSV (moi ∼3) was bound to the cells at 4°C followed by 1 h of internalization at 37°C. Cells were trypsinized, fixed and stained with anti-N-AF488 antibody, and the MFI of AF-488 measured by FACS.

    Article Snippet: Inhibitors, antibodies and plasmids The inhibitors used included: PI-103 (Alexis Biochemicals), dynasore, dyngo-4a,dynol-31-2, pitstop-2 (Ascent Scientific), pirl1 (Chembridge), wiskostatin (Enzo), CAS 879127-07-8, CAS 371942-96-7, dec-RVKR-CMK, LY294002, NSC23766, staurosporine, taxol, wortmannin, Y27632, α-PDX (Calbiochem), bafilomycin A, blebbistatin, calphostin C, chlorpromazine, cytochalasin D, EIPA, genistein, IPA-3, jasplakinolide, latrunculin A, leupeptin, ML7, monensin, NH4Cl, nocodazole, and rottlerin (Sigma).

    Techniques: Infection, Staining, Microscopy, Incubation, SDS Page, Western Blot, Indirect Immunoperoxidase Assay, FACS, Expressing

    Morphology of pollen tubes treated with increasing doses of latrunculin B or cytochalasin D. (A) Control with no treatment. (B–H) latrunculin B: (B) 1 nM, (C) 2 nM, (D) 3 nM, (E) 4 nM, (F) 8 nM, (G) 15 nM, (H) 25 nM; (I–L) cytochalasin D: (I) 200 nM (J) 400 nM, (K) 800 nM, (L) 1200 nM. Bar, 10 μm. Note the reduction of the clear zone in B; also note the dramatic changes in growth pattern in C and D. The morphology of the cells does not change much between 4 and 15 nM (E–G), but at 25 nM latrunculin B (H) and 1200 nM cytochalasin D (L), the central part of the apical region of the cell becomes smooth, loosing the granularity that characterizes the clear zone. Video files showing the streaming patterns of a control cell (A) and a 3 nM latrunculin B–treated cell (D) are linked to the left image from each pair. (Videos 1 and 2).

    Journal: Molecular Biology of the Cell

    Article Title: Actin Polymerization Is Essential for Pollen Tube Growth V⃞

    doi:

    Figure Lengend Snippet: Morphology of pollen tubes treated with increasing doses of latrunculin B or cytochalasin D. (A) Control with no treatment. (B–H) latrunculin B: (B) 1 nM, (C) 2 nM, (D) 3 nM, (E) 4 nM, (F) 8 nM, (G) 15 nM, (H) 25 nM; (I–L) cytochalasin D: (I) 200 nM (J) 400 nM, (K) 800 nM, (L) 1200 nM. Bar, 10 μm. Note the reduction of the clear zone in B; also note the dramatic changes in growth pattern in C and D. The morphology of the cells does not change much between 4 and 15 nM (E–G), but at 25 nM latrunculin B (H) and 1200 nM cytochalasin D (L), the central part of the apical region of the cell becomes smooth, loosing the granularity that characterizes the clear zone. Video files showing the streaming patterns of a control cell (A) and a 3 nM latrunculin B–treated cell (D) are linked to the left image from each pair. (Videos 1 and 2).

    Article Snippet: In previous studies in pollen tubes ( ; ) and root hairs , it has been observed that cytoplasmic streaming continues in cells that are not elongating after latrunculin B or cytochalasin D treatment, but no effort to quantitate these rates was made.

    Techniques:

    F-actin distribution of cells treated with 2 nM latrunculin B. Treated cells after chemical fixation and Alexa-phalloidin staining. (A) Control cells. (B) Cells treated with 2 nM latrunculin B for 20 min before fixation. F-actin was visualized by confocal microscopy; the images are projections of 30 optical sections at 0.5-μm intervals in the Z -axis. There is a video file with a 3-dimensional reconstruction and rotation linked to the first projection from each panel (Videos 3 and 4). Note the prominent subapical funnel-shaped F-actin rich region (arrow in A) that is absent in the treated cells. Bar, 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Actin Polymerization Is Essential for Pollen Tube Growth V⃞

    doi:

    Figure Lengend Snippet: F-actin distribution of cells treated with 2 nM latrunculin B. Treated cells after chemical fixation and Alexa-phalloidin staining. (A) Control cells. (B) Cells treated with 2 nM latrunculin B for 20 min before fixation. F-actin was visualized by confocal microscopy; the images are projections of 30 optical sections at 0.5-μm intervals in the Z -axis. There is a video file with a 3-dimensional reconstruction and rotation linked to the first projection from each panel (Videos 3 and 4). Note the prominent subapical funnel-shaped F-actin rich region (arrow in A) that is absent in the treated cells. Bar, 10 μm.

    Article Snippet: In previous studies in pollen tubes ( ; ) and root hairs , it has been observed that cytoplasmic streaming continues in cells that are not elongating after latrunculin B or cytochalasin D treatment, but no effort to quantitate these rates was made.

    Techniques: Staining, Confocal Microscopy

    Effect of latrunculin B on the oscillatory growth of the pollen tube. (A) Growth rate profile for 5 min of three representative control cells. (B) Growth rate profile of three representative cells treated with 1 nM latrunculin B. Note that although the treated cells are still growing, their oscillatory pattern has been disrupted.

    Journal: Molecular Biology of the Cell

    Article Title: Actin Polymerization Is Essential for Pollen Tube Growth V⃞

    doi:

    Figure Lengend Snippet: Effect of latrunculin B on the oscillatory growth of the pollen tube. (A) Growth rate profile for 5 min of three representative control cells. (B) Growth rate profile of three representative cells treated with 1 nM latrunculin B. Note that although the treated cells are still growing, their oscillatory pattern has been disrupted.

    Article Snippet: In previous studies in pollen tubes ( ; ) and root hairs , it has been observed that cytoplasmic streaming continues in cells that are not elongating after latrunculin B or cytochalasin D treatment, but no effort to quantitate these rates was made.

    Techniques:

    The effect of cytarabine alone compared with sorafenib plus cytarabine on leukemic cell colonization in the spleen and on spleen size. NOD-SCID-IL2R γ null mice were injected with luciferase-labeled U937 cells and treated 3 days later with cytarabine

    Journal: JNCI Journal of the National Cancer Institute

    Article Title: Activity of the Multikinase Inhibitor Sorafenib in Combination With Cytarabine in Acute Myeloid Leukemia

    doi: 10.1093/jnci/djr107

    Figure Lengend Snippet: The effect of cytarabine alone compared with sorafenib plus cytarabine on leukemic cell colonization in the spleen and on spleen size. NOD-SCID-IL2R γ null mice were injected with luciferase-labeled U937 cells and treated 3 days later with cytarabine

    Article Snippet: Cytarabine was purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Mouse Assay, Injection, Luciferase, Labeling

    Effect of Sorafenib on the Uptake and Accumulation of Cytarabine in AML Cell Lines

    Journal: JNCI Journal of the National Cancer Institute

    Article Title: Activity of the Multikinase Inhibitor Sorafenib in Combination With Cytarabine in Acute Myeloid Leukemia

    doi: 10.1093/jnci/djr107

    Figure Lengend Snippet: Effect of Sorafenib on the Uptake and Accumulation of Cytarabine in AML Cell Lines

    Article Snippet: Cytarabine was purchased from Sigma-Aldrich (St Louis, MO).

    Techniques:

    Antileukemic activity of sorafenib alone, cytarabine alone, and sorafenib plus cytarabine in a U937 xenograft model. NOD-SCID-IL2R γ null mice were injected with luciferase-labeled (Luc + ) U937 cells and treated 3 days later with sorafenib alone (60

    Journal: JNCI Journal of the National Cancer Institute

    Article Title: Activity of the Multikinase Inhibitor Sorafenib in Combination With Cytarabine in Acute Myeloid Leukemia

    doi: 10.1093/jnci/djr107

    Figure Lengend Snippet: Antileukemic activity of sorafenib alone, cytarabine alone, and sorafenib plus cytarabine in a U937 xenograft model. NOD-SCID-IL2R γ null mice were injected with luciferase-labeled (Luc + ) U937 cells and treated 3 days later with sorafenib alone (60

    Article Snippet: Cytarabine was purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Activity Assay, Mouse Assay, Injection, Luciferase, Labeling

    Sorafenib and Cytarabine Combination Treatment of AML Cell Lines and Primary Blast Samples

    Journal: JNCI Journal of the National Cancer Institute

    Article Title: Activity of the Multikinase Inhibitor Sorafenib in Combination With Cytarabine in Acute Myeloid Leukemia

    doi: 10.1093/jnci/djr107

    Figure Lengend Snippet: Sorafenib and Cytarabine Combination Treatment of AML Cell Lines and Primary Blast Samples

    Article Snippet: Cytarabine was purchased from Sigma-Aldrich (St Louis, MO).

    Techniques:

    Effect of sorafenib alone, cytarabine alone, and sorafenib plus cytarabine on cell viability in primary childhood acute myeloid leukemia blast samples. Freshly isolated primary leukemic blast cells were treated with A ) sorafenib (10 μM), B ) cytarabine

    Journal: JNCI Journal of the National Cancer Institute

    Article Title: Activity of the Multikinase Inhibitor Sorafenib in Combination With Cytarabine in Acute Myeloid Leukemia

    doi: 10.1093/jnci/djr107

    Figure Lengend Snippet: Effect of sorafenib alone, cytarabine alone, and sorafenib plus cytarabine on cell viability in primary childhood acute myeloid leukemia blast samples. Freshly isolated primary leukemic blast cells were treated with A ) sorafenib (10 μM), B ) cytarabine

    Article Snippet: Cytarabine was purchased from Sigma-Aldrich (St Louis, MO).

    Techniques: Isolation

    P2X 7 R mediates LL-37 internalization by human macrophages. ( A and B ) dTHP-1 cells were pretreated with P2X 7 R inhibitors KN-62 (1 μM) or oxATP (100 μM) for 1 h and incubated with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). (A) A representative FACS plot. ( C and D ) Control (transfected with nontarget shRNA) and P2X 7 R-KD (transfected with human P2X 7 R-targeted shRNA) dTHP-1 cells were incubated with 10 μg/ml of FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was analyzed by flow cytometry ( n = 5). (C) One representative FACS plot. ( E ) WT, control, and P2X 7 R-KD THP-1 cells were pretreated with P2X 7 R inhibitor KN-62 (1 μM) for 1 h before incubation with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( F ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and P2X 7 R was visualized using a confocal microscope. ( G ) Control and P2X 7 R-KD dTHP-1 cells were pretreated with nystatin (10 μg/ml) or dynasore (20 μM), respectively, at 37°C for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 5). ( H ) dTHP-1 cells were pretreated with BzATP (100 μM, specific agonist of P2X 7 R), (-)-blebbistatin (200 μM, inhibitor of nonmuscle myosin II), 10 PanX (1 μM, peptide antagonist of Panx-1), FIPI (1 μM, inhibitor of PLD isoforms), wortmannin (1 μM, specific inhibitor of PI 3 K), geldanamycin (1 μM, inhibitor of hsp90), U0126 (1 μM, inhibitor of ERK MAPK), or SB203580 (1 μM, inhibitor of p38 MAPK), followed by incubation with FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( I ) The colocalization of LL-37/P2X 7 R complex and caveolin-1 or clathrin was visualized using a confocal microscope. Images in (I a ) and (I b ) are enlargements of the boxed areas. A total of 10 μg/ml of LL-37 was used in all treatments, and the confocal images are representative of at least three experiments. Scale bars, 10 μm. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance

    doi: 10.4049/jimmunol.1402845

    Figure Lengend Snippet: P2X 7 R mediates LL-37 internalization by human macrophages. ( A and B ) dTHP-1 cells were pretreated with P2X 7 R inhibitors KN-62 (1 μM) or oxATP (100 μM) for 1 h and incubated with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). (A) A representative FACS plot. ( C and D ) Control (transfected with nontarget shRNA) and P2X 7 R-KD (transfected with human P2X 7 R-targeted shRNA) dTHP-1 cells were incubated with 10 μg/ml of FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was analyzed by flow cytometry ( n = 5). (C) One representative FACS plot. ( E ) WT, control, and P2X 7 R-KD THP-1 cells were pretreated with P2X 7 R inhibitor KN-62 (1 μM) for 1 h before incubation with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( F ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and P2X 7 R was visualized using a confocal microscope. ( G ) Control and P2X 7 R-KD dTHP-1 cells were pretreated with nystatin (10 μg/ml) or dynasore (20 μM), respectively, at 37°C for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 5). ( H ) dTHP-1 cells were pretreated with BzATP (100 μM, specific agonist of P2X 7 R), (-)-blebbistatin (200 μM, inhibitor of nonmuscle myosin II), 10 PanX (1 μM, peptide antagonist of Panx-1), FIPI (1 μM, inhibitor of PLD isoforms), wortmannin (1 μM, specific inhibitor of PI 3 K), geldanamycin (1 μM, inhibitor of hsp90), U0126 (1 μM, inhibitor of ERK MAPK), or SB203580 (1 μM, inhibitor of p38 MAPK), followed by incubation with FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( I ) The colocalization of LL-37/P2X 7 R complex and caveolin-1 or clathrin was visualized using a confocal microscope. Images in (I a ) and (I b ) are enlargements of the boxed areas. A total of 10 μg/ml of LL-37 was used in all treatments, and the confocal images are representative of at least three experiments. Scale bars, 10 μm. * p

    Article Snippet: PMA, 2-ME, HEPES, RPMI 1640 medium, cytochalasin B (CytoB), KN-62, oxidized ATP (oxATP), BzATP, filipin III, dynasore hydrate, nystatin, (-)-blebbistatin, wortmannin, geldanamycin, chloropromazine (CLQ), chloroquine (CLQ), FIPI hydrochloride hydrate, RIPA buffer, RPMI 1640 medium, and LPS (from Salmonella enterica ) were from Sigma-Aldrich (St. Louis, MO); U0126 and SB203580 were purchased from Tocris Bioscience (Bristol, U.K.); fluorescein-conjugated Staphylococcus aureus , FBS, and M-CSF were purchased from Life Technologies (Paisley, U.K.); Abs against clathrin, caveolin-1, and P2X7 R were from Santa Cruz Biotechnology (Santa Cruz, CA); and synthetic LL-37 (NH2 -LLGDFFRKSKEKIGKEFKRIVQRIKDFFRNLVPRTES-COOH), FAM- or TAMRA-conjugated LL-37, TAMRA-conjugated sequence-scrambled LL-37 (sLL-37;GLKLRFEFSKIKGEFLKTPEVRFRDIKLKDNRISVQR), and Panx-1 antagonist peptide 10 Panx (WRQAAFVDSY) were from Innovagen (Lund, Sweden).

    Techniques: Incubation, Labeling, Flow Cytometry, Cytometry, FACS, Transfection, shRNA, Microscopy

    Knockdown of TNFAIP8 induces epithelial ovarian cancer cell cycle arrest. (A) Following siNC or siTNFAIP8 transfection for 48 h, A2780s or A2780cp cells were collected for propidium iodide staining, followed by flow cytometry analysis. The percentage of cells in each stage was analyzed. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: TNFAIP8 promotes cell growth by regulating the Hippo pathway in epithelial ovarian cancer

    doi: 10.3892/etm.2018.6819

    Figure Lengend Snippet: Knockdown of TNFAIP8 induces epithelial ovarian cancer cell cycle arrest. (A) Following siNC or siTNFAIP8 transfection for 48 h, A2780s or A2780cp cells were collected for propidium iodide staining, followed by flow cytometry analysis. The percentage of cells in each stage was analyzed. *P

    Article Snippet: Cells were fixed in 70% ethanol at 4°C for 15 min, washed in PBS, incubated in 50 µg/ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) containing 0.1 mg/ml RNase A (Sigma-Aldrich; Merck KGaA) and 0.1% Triton X (Sigma-Aldrich; Merck KGaA) for 30 min and were analyzed on a Cytomics FC500 flow cytometer (Beckman Coulter, Inc.).

    Techniques: Transfection, Staining, Flow Cytometry, Cytometry

    Localization of F-actin in MCF-7/MR cells and inhibition of actin polymerization by CytD. (A) Cells grown for 7 days on glass cover slips were fixed, permeabilized with Triton-X-100 and reacted with anti-ZO-1 antibody (a and d) and with rhodamine phalloidin (b and e) to follow F-actin. The localization of actin and ZO-1 at the EVs surface is shown for EVs formed between two (a–c) and/or multiple attached cells (d–f). Arrows denote the localization of cell-cell attachment zones (belt-like structures). (B) Cells were either untreated (a–c) or treated with CytD (10 µg/ml) for 30 min at 37°C (d–f), washed and reacted as in panel A, to visualize ZO-1 (a and d) and F-actin (b and e). (C) Cells were treated as described in panel B and then stained for ABCG2 (a and d) and F-actin (b and e). (D) Cells were grown in riboflavin (B2)-deficient medium for 48 hr prior to CytD treatment to avoid riboflavin accumulation in EVs. Cells were then washed and transferred to riboflavin-containing medium for an additional 24 hr to examine the riboflavin accumulation capacity (a–c). Untreated cells grown continuously in medium containing (d–f) or lacking riboflavin (g–i) served as controls. Arrows denote the location of EVs. Cells were analyzed using a Zeiss inverted Cell-Observer microscope at a magnification of ×630 (A–C) or ×200 (D). The merged images including DAPI staining (panels A–C), were generated using Cell-Observer software.

    Journal: PLoS ONE

    Article Title: Structure and Function of ABCG2-Rich Extracellular Vesicles Mediating Multidrug Resistance

    doi: 10.1371/journal.pone.0016007

    Figure Lengend Snippet: Localization of F-actin in MCF-7/MR cells and inhibition of actin polymerization by CytD. (A) Cells grown for 7 days on glass cover slips were fixed, permeabilized with Triton-X-100 and reacted with anti-ZO-1 antibody (a and d) and with rhodamine phalloidin (b and e) to follow F-actin. The localization of actin and ZO-1 at the EVs surface is shown for EVs formed between two (a–c) and/or multiple attached cells (d–f). Arrows denote the localization of cell-cell attachment zones (belt-like structures). (B) Cells were either untreated (a–c) or treated with CytD (10 µg/ml) for 30 min at 37°C (d–f), washed and reacted as in panel A, to visualize ZO-1 (a and d) and F-actin (b and e). (C) Cells were treated as described in panel B and then stained for ABCG2 (a and d) and F-actin (b and e). (D) Cells were grown in riboflavin (B2)-deficient medium for 48 hr prior to CytD treatment to avoid riboflavin accumulation in EVs. Cells were then washed and transferred to riboflavin-containing medium for an additional 24 hr to examine the riboflavin accumulation capacity (a–c). Untreated cells grown continuously in medium containing (d–f) or lacking riboflavin (g–i) served as controls. Arrows denote the location of EVs. Cells were analyzed using a Zeiss inverted Cell-Observer microscope at a magnification of ×630 (A–C) or ×200 (D). The merged images including DAPI staining (panels A–C), were generated using Cell-Observer software.

    Article Snippet: Chemicals Cytochalasin D (CytD), nocodazole, Hoechst 33342, fumitremorgin C (FTC), MR, topotecan, riboflavin, DAB, 3′-amino 9′-ethyl carbazole (AEC), hematoxylin, Triton X-100 and DAPI were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Inhibition, Cell Attachment Assay, Staining, Microscopy, Generated, Software

    PI3K inhibition reduced morphine-induced microglial migration. Primary microglial cells were treated with two PI3K inhibitors, wortmannin and LY294002, for 1 h then treated for 2 h with morphine and allowed to migrate toward 10 μ m ADP. Migrated

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Morphine Enhances Microglial Migration through Modulation of P2X4 Receptor Signaling

    doi: 10.1523/JNEUROSCI.4595-08.2009

    Figure Lengend Snippet: PI3K inhibition reduced morphine-induced microglial migration. Primary microglial cells were treated with two PI3K inhibitors, wortmannin and LY294002, for 1 h then treated for 2 h with morphine and allowed to migrate toward 10 μ m ADP. Migrated

    Article Snippet: Cells were treated with antagonists [ d -Phe-Cys-Tyr- d -Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), 2′,3′- O -(2,4,6-trinitrophenyl)-ATP (TNP-ATP), wortmannin, or LY294002, Sigma] for 1 h, then stimulated with agonists [morphine, [ d -Ala2 , N -Me-Phe4 , Gly5 -ol]-enkephalin (DAMGO), [ d -Pen2,5 ]-enkephalin (DPDPE), or U-69593, Sigma)] for 2, 6, 12, 24, or 48 h. Cells were counted post-treatment with trypan blue to insure survival ( > 99% viability), then added (100 × 103 cells in 100 μ l) to the top chamber of a transwell plate with fibronectin-coated membranes and 500 μ l of 10 μ m ADP in the bottom well.

    Techniques: Inhibition, Migration

    Intracellular signaling mediates adhesion to anti-TCR treated plates. (A) Jurkat E6.1 cells were stimulated with various concentrations of the immobilized anti-TCR antibodies OKT3 and IP26 for 30 minutes. Total changes in TCR-inducible tyrosine phosphorylation were detected by immunoblot (left panel). TCR-induced adhesion in response to these antibodies or two non-specific antibodies (IgG2a and total mouse IgG) was also quantified and is graphed as the average three experiments ± SD (right panel). These data are shown as the average of three experiments (B) Jurkat E6.1 cells (left panel) and hAPBTs (right panel) were stained and pretreated with DMSO or the actin cytoskeleton inhibitors Cytochalasin D (Cyto D) and Latrunculin B (Lat B) for 30 minutes or jasplakinolide (Jasp) for 15 minutes at 37°C. The cells were applied to an anti-TCR coated RIA/EIA plate and allowed to adhere for 30 minutes at 37°C. The data were normalized such that the DMSO samples were set to 100%. The normalized results from three independent experiments were then graphed. ** p≤0.01 *** p≤0.001.

    Journal: PLoS ONE

    Article Title: Non-Catalytic Functions of Pyk2 and Fyn Regulate Late Stage Adhesion in Human T Cells

    doi: 10.1371/journal.pone.0053011

    Figure Lengend Snippet: Intracellular signaling mediates adhesion to anti-TCR treated plates. (A) Jurkat E6.1 cells were stimulated with various concentrations of the immobilized anti-TCR antibodies OKT3 and IP26 for 30 minutes. Total changes in TCR-inducible tyrosine phosphorylation were detected by immunoblot (left panel). TCR-induced adhesion in response to these antibodies or two non-specific antibodies (IgG2a and total mouse IgG) was also quantified and is graphed as the average three experiments ± SD (right panel). These data are shown as the average of three experiments (B) Jurkat E6.1 cells (left panel) and hAPBTs (right panel) were stained and pretreated with DMSO or the actin cytoskeleton inhibitors Cytochalasin D (Cyto D) and Latrunculin B (Lat B) for 30 minutes or jasplakinolide (Jasp) for 15 minutes at 37°C. The cells were applied to an anti-TCR coated RIA/EIA plate and allowed to adhere for 30 minutes at 37°C. The data were normalized such that the DMSO samples were set to 100%. The normalized results from three independent experiments were then graphed. ** p≤0.01 *** p≤0.001.

    Article Snippet: Cytochalasin D, jasplakinolide, and PP2 were obtained from Calbiochem.

    Techniques: Staining, Enzyme-linked Immunosorbent Assay

    The actin cytoskeleton is necessary for late TCR-induced adhesion. (A) DMSO, Cytochalasin D (Cyto D), Latrunculin B (Lat B), or jasplakinolide (Jasp) was added to the stained Jurkat cells 15 minutes after beginning TCR stimulation on RIA/EIA plates for 30 minutes. The data were normalized to the DMSO controls, and the average of three independent experiments ± SD is depicted. * p≤0.05 *** p≤0.001(B) Jurkat E6.1 cells or hAPBTs were stained and treated with DMSO or the microtubule depolymerizing chemical colchicine for 30 minutes at 37°C. The DMSO samples were set to 100% and the average normalized data from three independent replicates ± SD was graphed. * p≤0.05 (C) Jurkat E6.1 cells or hAPBTs were treated and stimulated as in (B) but with the microtubule stabilizing agent paclitaxel. The normalized values from three independent replicates ± SD are shown graphically. N.D. demonstrates that no data were collected for this dose in hAPBTs.

    Journal: PLoS ONE

    Article Title: Non-Catalytic Functions of Pyk2 and Fyn Regulate Late Stage Adhesion in Human T Cells

    doi: 10.1371/journal.pone.0053011

    Figure Lengend Snippet: The actin cytoskeleton is necessary for late TCR-induced adhesion. (A) DMSO, Cytochalasin D (Cyto D), Latrunculin B (Lat B), or jasplakinolide (Jasp) was added to the stained Jurkat cells 15 minutes after beginning TCR stimulation on RIA/EIA plates for 30 minutes. The data were normalized to the DMSO controls, and the average of three independent experiments ± SD is depicted. * p≤0.05 *** p≤0.001(B) Jurkat E6.1 cells or hAPBTs were stained and treated with DMSO or the microtubule depolymerizing chemical colchicine for 30 minutes at 37°C. The DMSO samples were set to 100% and the average normalized data from three independent replicates ± SD was graphed. * p≤0.05 (C) Jurkat E6.1 cells or hAPBTs were treated and stimulated as in (B) but with the microtubule stabilizing agent paclitaxel. The normalized values from three independent replicates ± SD are shown graphically. N.D. demonstrates that no data were collected for this dose in hAPBTs.

    Article Snippet: Cytochalasin D, jasplakinolide, and PP2 were obtained from Calbiochem.

    Techniques: Staining, Enzyme-linked Immunosorbent Assay

    T cell morphology and actin structure differ between the early and late stages of TCR activation. (A) Jurkat cells were pre-treated with DMSO or jasplakinolide (Jasp) for 15 minutes at 37°C. These cells were then stimulated with or without plate-bound anti-TCR for the indicated times. Fractions containing G-actin and F-actin were isolated and then run on a polyacrylimide gel. Immunoblotting was used to detect changes in G-actin and F-actin. A representative blot is shown (top panel). The ratio of F-actin to G-actin was determined, and the average ratio from three independent experiments ± SEM was calculated and graphed (bottom panel). (B) Jurkat cells were stimulated with anti-TCR on plastic chamber slides for the indicated times. These cells were then stained using FITC-phallodin and imaged using epifluorescence. The phallodin images shown at each time point are representative of three experiments. Yellow scale bar = 10 µM. (C) The average blinded score of thirty individual cells at each time was determined and graphed. Examples of each scoring category are shown in the key below the graph.

    Journal: PLoS ONE

    Article Title: Non-Catalytic Functions of Pyk2 and Fyn Regulate Late Stage Adhesion in Human T Cells

    doi: 10.1371/journal.pone.0053011

    Figure Lengend Snippet: T cell morphology and actin structure differ between the early and late stages of TCR activation. (A) Jurkat cells were pre-treated with DMSO or jasplakinolide (Jasp) for 15 minutes at 37°C. These cells were then stimulated with or without plate-bound anti-TCR for the indicated times. Fractions containing G-actin and F-actin were isolated and then run on a polyacrylimide gel. Immunoblotting was used to detect changes in G-actin and F-actin. A representative blot is shown (top panel). The ratio of F-actin to G-actin was determined, and the average ratio from three independent experiments ± SEM was calculated and graphed (bottom panel). (B) Jurkat cells were stimulated with anti-TCR on plastic chamber slides for the indicated times. These cells were then stained using FITC-phallodin and imaged using epifluorescence. The phallodin images shown at each time point are representative of three experiments. Yellow scale bar = 10 µM. (C) The average blinded score of thirty individual cells at each time was determined and graphed. Examples of each scoring category are shown in the key below the graph.

    Article Snippet: Cytochalasin D, jasplakinolide, and PP2 were obtained from Calbiochem.

    Techniques: Activation Assay, Isolation, Staining

    Effect of Paclitaxel ( Pac ) on cell deformation. HL‐60 cells were incubated with varying concentrations of Pac and cell deformation was observed using RT‐DC. (a) Scatter plots of HL‐60 cell populations incubated without (Control) and with 1 μM Pac . The contour lines compare the 50% event density of the control (black) vs Pac (green). The inset shows the reservoir measurement for reference. (b) Representative fluorescence images (microtubule, green and DNA, blue). Scale bar is 10 μm. (c) Dose–response graph showing mean RD values of three replicates of HL‐60 cell population treated with increasing concentrations of Pac that allowed for the extraction of a half‐maximal concentration (EC 50 ) for the effect of Pac on the deformation of HL‐60 cells. Shaded red area indicates 95% confidence interval of the fit. Tangential slope at the inflection point of the sigmoidal fit function is 0.3 with Hill Coefficient 1.61. Error bars are standard error of the mean (SEM). All experiments were carried out at a flow rate of 0.04 μL·s −1 in a 20 × 20 × 300 μm (w × h × l) channel. p values denote *** p ≤ 0.001 and ns, not significant. [Color figure can be viewed at wileyonlinelibrary.com ]

    Journal: Cytoskeleton (Hoboken, N.j.)

    Article Title: High‐throughput cell mechanical phenotyping for label‐free titration assays of cytoskeletal modifications. High‐throughput cell mechanical phenotyping for label‐free titration assays of cytoskeletal modifications

    doi: 10.1002/cm.21369

    Figure Lengend Snippet: Effect of Paclitaxel ( Pac ) on cell deformation. HL‐60 cells were incubated with varying concentrations of Pac and cell deformation was observed using RT‐DC. (a) Scatter plots of HL‐60 cell populations incubated without (Control) and with 1 μM Pac . The contour lines compare the 50% event density of the control (black) vs Pac (green). The inset shows the reservoir measurement for reference. (b) Representative fluorescence images (microtubule, green and DNA, blue). Scale bar is 10 μm. (c) Dose–response graph showing mean RD values of three replicates of HL‐60 cell population treated with increasing concentrations of Pac that allowed for the extraction of a half‐maximal concentration (EC 50 ) for the effect of Pac on the deformation of HL‐60 cells. Shaded red area indicates 95% confidence interval of the fit. Tangential slope at the inflection point of the sigmoidal fit function is 0.3 with Hill Coefficient 1.61. Error bars are standard error of the mean (SEM). All experiments were carried out at a flow rate of 0.04 μL·s −1 in a 20 × 20 × 300 μm (w × h × l) channel. p values denote *** p ≤ 0.001 and ns, not significant. [Color figure can be viewed at wileyonlinelibrary.com ]

    Article Snippet: 4.3 Drug treatments HL‐60 cells were treated with different concentrations of Cytochalasin D , Jasplakinolide , Nocodazole , Paclitaxel , and Trichostatin A (all purchased from Sigma Aldrich, Germany).

    Techniques: Incubation, Fluorescence, Concentration Assay, Flow Cytometry

    Entry of RVFV ns depends on vacuolar acidification. (A) BHK-21 or A549 cells were pretreated for 1 h with different concentrations of bafilomycin A1 or ammonium chloride (NH 4 Cl) and subsequently infected with RVFV ns (MOI of ∼0.1) or VSV ns (MOI

    Journal: Journal of Virology

    Article Title: Acid-Activated Structural Reorganization of the Rift Valley Fever Virus Gc Fusion Protein

    doi: 10.1128/JVI.01973-12

    Figure Lengend Snippet: Entry of RVFV ns depends on vacuolar acidification. (A) BHK-21 or A549 cells were pretreated for 1 h with different concentrations of bafilomycin A1 or ammonium chloride (NH 4 Cl) and subsequently infected with RVFV ns (MOI of ∼0.1) or VSV ns (MOI

    Article Snippet: Bafilomycin A1, dynasore, cytochalasin D, nystatin (Sigma), and dyngo-4a (Abcam) were prepared in dimethyl sulfoxide (DMSO).

    Techniques: Infection

    Representative chromatograms and UV–vis absorption spectra showing the complexation of metal ions and reduction of cytochrome c. Panel (A) shows the complexation of Pb by sEPS1 (alginate and BSA) on the anode (orange), the complexation of Fe by sEPS1 on the cathode (blue), and the complexation of Pb when Cyto. c (alginate, BSA, and cytochrome c) was on the anode (red). Panel (B) shows the contrast of UV–vis absorption spectra from cytochrome c before and after corrosion tests on the anode and cathode in which anaerobic or aerobic states the condition on the cathode side and those after the colon indicate the location of Cyto. c.

    Journal: ACS Omega

    Article Title: Understanding the Impact of Extracellular Polymeric Substances on Lead Release in Drinking Water Systems

    doi: 10.1021/acsomega.8b02363

    Figure Lengend Snippet: Representative chromatograms and UV–vis absorption spectra showing the complexation of metal ions and reduction of cytochrome c. Panel (A) shows the complexation of Pb by sEPS1 (alginate and BSA) on the anode (orange), the complexation of Fe by sEPS1 on the cathode (blue), and the complexation of Pb when Cyto. c (alginate, BSA, and cytochrome c) was on the anode (red). Panel (B) shows the contrast of UV–vis absorption spectra from cytochrome c before and after corrosion tests on the anode and cathode in which anaerobic or aerobic states the condition on the cathode side and those after the colon indicate the location of Cyto. c.

    Article Snippet: Within each group, three different sEPS conditions were examined: 100 mg/L alginate + 100 mg/L BSA (labeled sEPS1); 200 mg/L alginate + 200 mg/L BSA (labeled sEPS2); and 100 mg/L alginate + 100 mg/L BSA + 123.84 mg/L cytochrome c (C7752, Sigma-Aldrich) (labeled Cyto. c).

    Techniques:

    Numbers of circulating endothelial cells (defined as CD45 NEG , CD146 POS , CD133 NEG and DAPI bright with viable nuclei), with Day 1 draw immediately prior to initiation of first cycle of treatment and the final draw at time of disease progression.

    Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

    Article Title: Phase II study of cediranib (AZD 2171), an inhibitor of the vascular endothelial growth factor receptor, for second-line therapy of small cell lung cancer (NCI # 7097)

    doi: 10.1097/JTO.0b013e3181e2fcb0

    Figure Lengend Snippet: Numbers of circulating endothelial cells (defined as CD45 NEG , CD146 POS , CD133 NEG and DAPI bright with viable nuclei), with Day 1 draw immediately prior to initiation of first cycle of treatment and the final draw at time of disease progression.

    Article Snippet: After washing, slides were incubated with FITC-conjugated anti-CD146-antibody (cat. # MAB16985F, Chemicon International, Inc.), PE-conjugated anti-CD133-antibody (cat. # 130-080-801, Miltenyi Biotec) and counterstained with DAPI (Sigma).

    Techniques:

    Neutralizing effect of mAb 286, mapping of its binding site, and analysis of binding to VEGF-D variants with mutated residues in N-terminal α-helix. A , the capacity of mAb 286 to block binding and cross-linking, by VEGF-DΔNΔC, of chimeric receptors containing VEGFR-2 ( left ) or VEGFR-3 ( right ) extracellular domains was assessed in bioassays (see “Experimental Procedures”). Also included were neutralizing mAb VD1, which binds loop 2 of VEGF-DΔNΔC, and mAb VD4, which binds, but does not neutralize, VEGF-DΔNΔC ( 39 ). B , peptide-based mapping of the mAb 286 binding site in VEGF-DΔNΔC by ELISA (see “Experimental Procedures”). The ratio of signal to background for the interaction of mAb 286 with immobilized peptides is shown on the y axis of the graph, and the x axis indicates the identifier numbers of peptides. Top box above the graph , amino acid sequence for the VEGF homology domain of human VEGF-D; N-terminal residue (phenylalanine) is number 89, and the C-terminal residue (arginine) is 205. Bottom box above the graph , examples of peptides used in mapping (mAb 286 binding site is in a rectangle ). The FLAG sequence is shown in boldface type in peptide 36, which lacks the VEGF-D-derived sequence, and was the negative control. C , detection of VEGF-DΔNΔC variants by Western blotting under reducing and denaturing conditions using mAb 286 ( top ) or M2 anti-FLAG mAb as a positive control ( bottom ). Each well contained 30 ng of purified protein. VEGF-D , VEGF-DΔNΔC; variants of this protein each have one residue mutated to alanine, as indicated. Positions of molecular mass markers (in kDa) are shown to the left . The histogram under the blots shows intensities of bands for VEGF-D variants (mean ± S.D.) relative to the intensity of the band for VEGF-DΔNΔC, as determined from Western blots with mAb 286. D , analysis of mAb 286 binding to VEGF-DΔNΔC variants by ELISA. M2 was used for capture and mAb 286 for detection; the y axis shows binding of variant proteins compared with VEGF-DΔNΔC (the latter defined as 100% binding), and the x axis lists VEGF-D variants. Equal amounts of VEGF-DΔNΔC and variants were used. For A , B , and D , assays were conducted three times. Columns , mean; error bars , S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D *

    doi: 10.1074/jbc.M116.736801

    Figure Lengend Snippet: Neutralizing effect of mAb 286, mapping of its binding site, and analysis of binding to VEGF-D variants with mutated residues in N-terminal α-helix. A , the capacity of mAb 286 to block binding and cross-linking, by VEGF-DΔNΔC, of chimeric receptors containing VEGFR-2 ( left ) or VEGFR-3 ( right ) extracellular domains was assessed in bioassays (see “Experimental Procedures”). Also included were neutralizing mAb VD1, which binds loop 2 of VEGF-DΔNΔC, and mAb VD4, which binds, but does not neutralize, VEGF-DΔNΔC ( 39 ). B , peptide-based mapping of the mAb 286 binding site in VEGF-DΔNΔC by ELISA (see “Experimental Procedures”). The ratio of signal to background for the interaction of mAb 286 with immobilized peptides is shown on the y axis of the graph, and the x axis indicates the identifier numbers of peptides. Top box above the graph , amino acid sequence for the VEGF homology domain of human VEGF-D; N-terminal residue (phenylalanine) is number 89, and the C-terminal residue (arginine) is 205. Bottom box above the graph , examples of peptides used in mapping (mAb 286 binding site is in a rectangle ). The FLAG sequence is shown in boldface type in peptide 36, which lacks the VEGF-D-derived sequence, and was the negative control. C , detection of VEGF-DΔNΔC variants by Western blotting under reducing and denaturing conditions using mAb 286 ( top ) or M2 anti-FLAG mAb as a positive control ( bottom ). Each well contained 30 ng of purified protein. VEGF-D , VEGF-DΔNΔC; variants of this protein each have one residue mutated to alanine, as indicated. Positions of molecular mass markers (in kDa) are shown to the left . The histogram under the blots shows intensities of bands for VEGF-D variants (mean ± S.D.) relative to the intensity of the band for VEGF-DΔNΔC, as determined from Western blots with mAb 286. D , analysis of mAb 286 binding to VEGF-DΔNΔC variants by ELISA. M2 was used for capture and mAb 286 for detection; the y axis shows binding of variant proteins compared with VEGF-DΔNΔC (the latter defined as 100% binding), and the x axis lists VEGF-D variants. Equal amounts of VEGF-DΔNΔC and variants were used. For A , B , and D , assays were conducted three times. Columns , mean; error bars , S.D.

    Article Snippet: Equal volumes of conditioned medium containing VEGF-DΔNΔC variants that were not tagged with the FLAG peptide were concentrated to the same final volume and buffer-exchanged into PBS using an Amicon size exclusion centrifugal filter with a 10 kDa nominal molecular mass limit (Millipore, Billerica, MA).

    Techniques: Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Derivative Assay, Negative Control, Western Blot, Positive Control, Purification, Variant Assay

    Receptor binding and activation by untagged VEGF-D variants. A , bioassays for binding and cross-linking of extracellular domains of VEGFR-2 ( left ) and VEGFR-3 ( right ) with altered versions of VEGF-DΔNΔC, Y94A, K100A, and I102A lacking FLAG tag. The same amount of each VEGF-DΔNΔC variant was used. Results are expressed as a percentage of fluorescence units generated relative to untagged VEGF-DΔNΔC ( y axis). VEGF-D , untagged form of VEGF-DΔNΔC. Assays were conducted three times. Columns , mean; error bars , S.D. *, statistically significant differences as assessed by one-way analysis of variance with Tukey's post hoc test. B , adult LECs were stimulated with matched quantities of untagged variants or left unstimulated ( No GF ). Lysates were immunoprecipitated ( IP ) with antibody against VEGFR-2 ( left ) or VEGFR-3 ( right ) and analyzed by reducing SDS-PAGE and Western blotting with antibody against phosphotyrosine ( pY ) to assess receptor activation ( top blots ) or with antibody against VEGFR-2 ( bottom left blot ) or VEGFR-3 ( bottom right blot ) to confirm the presence of each receptor. Sizes of molecular mass markers (in kDa) are shown to the left of the panels .

    Journal: The Journal of Biological Chemistry

    Article Title: Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D *

    doi: 10.1074/jbc.M116.736801

    Figure Lengend Snippet: Receptor binding and activation by untagged VEGF-D variants. A , bioassays for binding and cross-linking of extracellular domains of VEGFR-2 ( left ) and VEGFR-3 ( right ) with altered versions of VEGF-DΔNΔC, Y94A, K100A, and I102A lacking FLAG tag. The same amount of each VEGF-DΔNΔC variant was used. Results are expressed as a percentage of fluorescence units generated relative to untagged VEGF-DΔNΔC ( y axis). VEGF-D , untagged form of VEGF-DΔNΔC. Assays were conducted three times. Columns , mean; error bars , S.D. *, statistically significant differences as assessed by one-way analysis of variance with Tukey's post hoc test. B , adult LECs were stimulated with matched quantities of untagged variants or left unstimulated ( No GF ). Lysates were immunoprecipitated ( IP ) with antibody against VEGFR-2 ( left ) or VEGFR-3 ( right ) and analyzed by reducing SDS-PAGE and Western blotting with antibody against phosphotyrosine ( pY ) to assess receptor activation ( top blots ) or with antibody against VEGFR-2 ( bottom left blot ) or VEGFR-3 ( bottom right blot ) to confirm the presence of each receptor. Sizes of molecular mass markers (in kDa) are shown to the left of the panels .

    Article Snippet: Equal volumes of conditioned medium containing VEGF-DΔNΔC variants that were not tagged with the FLAG peptide were concentrated to the same final volume and buffer-exchanged into PBS using an Amicon size exclusion centrifugal filter with a 10 kDa nominal molecular mass limit (Millipore, Billerica, MA).

    Techniques: Binding Assay, Activation Assay, FLAG-tag, Variant Assay, Fluorescence, Generated, Immunoprecipitation, SDS Page, Western Blot

    HDACi promote DNA damage by DSBs formation in glioma cell lines. ( a ) Analysis of DNA DSB formation by immunofluorescence of γH2AX-positive nuclei in glioma cells treated for 48 h with 10 mM VPA, 10 μ M SAHA or 100 μ M TMZ. Data are mean±S.E.M. from five independent experiments. ( b ) WST-1 cell viability assay on glioma cell lines exposed to increasing concentrations of TMZ (from 10 to 500 μ M) for 48 h. Data shown are mean±S.E.M. from three independent experiments. ( c ) Analysis of γH2AX-positive nuclei on glioma cells treated with 5 μ M Q-VD-OPh, 10 μ M SAHA or both combined for 48 h. Bars depict mean±S.E.M. from four independent experiments. ( d ) ROS production measured by flow cytometry on glioma cells treated with 10 mM VPA, 10 μ M SAHA or with 10 μ M SAHA in the presence of 10 mM GSH. Data are mean±S.E.M. from four independent experiments. ( e ) Analysis of γH2AX-positive nuclei on glioma cells treated with 10 μ M SAHA or 100 μ M TMZ in the presence or absence of 15 mM N -acetyl- L -cysteine (NAC). Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed by the Student's T -Test, * P

    Journal: Cell Death & Disease

    Article Title: Histone deacetylase inhibitors promote glioma cell death by G2 checkpoint abrogation leading to mitotic catastrophe

    doi: 10.1038/cddis.2014.412

    Figure Lengend Snippet: HDACi promote DNA damage by DSBs formation in glioma cell lines. ( a ) Analysis of DNA DSB formation by immunofluorescence of γH2AX-positive nuclei in glioma cells treated for 48 h with 10 mM VPA, 10 μ M SAHA or 100 μ M TMZ. Data are mean±S.E.M. from five independent experiments. ( b ) WST-1 cell viability assay on glioma cell lines exposed to increasing concentrations of TMZ (from 10 to 500 μ M) for 48 h. Data shown are mean±S.E.M. from three independent experiments. ( c ) Analysis of γH2AX-positive nuclei on glioma cells treated with 5 μ M Q-VD-OPh, 10 μ M SAHA or both combined for 48 h. Bars depict mean±S.E.M. from four independent experiments. ( d ) ROS production measured by flow cytometry on glioma cells treated with 10 mM VPA, 10 μ M SAHA or with 10 μ M SAHA in the presence of 10 mM GSH. Data are mean±S.E.M. from four independent experiments. ( e ) Analysis of γH2AX-positive nuclei on glioma cells treated with 10 μ M SAHA or 100 μ M TMZ in the presence or absence of 15 mM N -acetyl- L -cysteine (NAC). Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed by the Student's T -Test, * P

    Article Snippet: Temozolomide (3,4-dihydro-3-methyl-4-oxoimidazo-[5,1-d]-1,2,3,5-tetrazine-8-carboxamide, TMZ), valproic acid (VPA), SAHA (suberanilohydroxamic acid, vorinostat), L- reduced glutathione (GSH) and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Immunofluorescence, Viability Assay, Flow Cytometry, Cytometry

    Timeline (from top to bottom) for neuronal differentiation of mouse embryonic stem cells (ES cells) into an expandable population of cerebellar neurons. Graphs show the stem cell stage (top panel: ES stage), the induction stage of embryoid bodies (EB) in SFEBq culture (EB differentiation stage), an intermediate expansion or proliferation stage of neural progenitor cells (NPCs) through stable passaging on laminin in NPCs medium (NPC expansion stage), and finally the maturation in vitro in NS21 medium ( in vitro maturation stage) and maturation in adult wild type mouse cerebella (bottom panel: in vivo maturation stage). P2 indicates passage 2 (of expansion stage); Arac indicates cytosine β-D-arabinofuranoside; Scale bars indicate 100 μm.

    Journal: Scientific Reports

    Article Title: An expandable embryonic stem cell-derived Purkinje neuron progenitor population that exhibits in vivo maturation in the adult mouse cerebellum

    doi: 10.1038/s41598-017-09348-1

    Figure Lengend Snippet: Timeline (from top to bottom) for neuronal differentiation of mouse embryonic stem cells (ES cells) into an expandable population of cerebellar neurons. Graphs show the stem cell stage (top panel: ES stage), the induction stage of embryoid bodies (EB) in SFEBq culture (EB differentiation stage), an intermediate expansion or proliferation stage of neural progenitor cells (NPCs) through stable passaging on laminin in NPCs medium (NPC expansion stage), and finally the maturation in vitro in NS21 medium ( in vitro maturation stage) and maturation in adult wild type mouse cerebella (bottom panel: in vivo maturation stage). P2 indicates passage 2 (of expansion stage); Arac indicates cytosine β-D-arabinofuranoside; Scale bars indicate 100 μm.

    Article Snippet: Next, neurons were refreshed (600 µl) with NS21 medium supplemented with cytosine β-D-arabinofuranoside (4 µM, Ara-C, Sigma-Aldrich) every 48 hrs.

    Techniques: Passaging, In Vitro, In Vivo

    Infection with live tachyzoites is not required for TLR11-mediated IL-12 production from DC. A , sort-purified CD11c + splenic DCs were incubated with YFP-expressing tachyzoites alone or in the presence of the indicated concentrations of cytochalasin D for 20 h. The percentage of infected cells (CD11c + YFP + ) was assayed by flow cytometry gated on DCs. B , amount of IL-12/23p40 present in supernatants of DCs co-cultured with tachyzoites (as described in A ) was measured by ELISA. The data shown are representative of six experiments. C , CD11c + splenic DCs were incubated with YFP-expressing tachyzoites (2:1 ratio) for 20 h, and brefeldin A was added during the last 6 h of the culture. IL-12 production was analyzed by intracellular cytokine staining. As a positive control, DCs were stimulated with recombinant T. gondii profilin (1 μg/ml) as described above. D , mice were infected intraperitoneally with 10 5 YFP-expressing tachyzoites. Twenty hours later spleen was isolated and the percentage of IL-12 producing and infected cells (YFP + ) was analyzed by intracellular cytokine staining after incubation of splenocytes for 6 h in the presence of brefeldin A. E , splenocytes depleted of DCs ( Sp ) were placed in the bottom of a Transwell plate (separating membrane: 0.4-μm pores) and stimulated with live parental profilin-sufficient strain tachyzoites ( Toxo , ΔTgPRFe/TgPRFi strain), recombinant profilin (1 μg/ml), or profilin-deficient strain tachyzoites ( TgPRF KO Toxo , ΔTgPRFe/TgPRFi strain+ATc) in the presence or absence of DCs (upper portion of Transwell plate). As a positive control, DCs were cultivated with tachyzoites or recombinant profilin without a separating membrane. IL-12/23p40 present in supernatants was measured by ELISA. The data shown are representative of three performed experiments. Error bars , S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: UNC93B1 Is Essential for TLR11 Activation and IL-12-dependent Host Resistance to Toxoplasma gondii *

    doi: 10.1074/jbc.M110.171025

    Figure Lengend Snippet: Infection with live tachyzoites is not required for TLR11-mediated IL-12 production from DC. A , sort-purified CD11c + splenic DCs were incubated with YFP-expressing tachyzoites alone or in the presence of the indicated concentrations of cytochalasin D for 20 h. The percentage of infected cells (CD11c + YFP + ) was assayed by flow cytometry gated on DCs. B , amount of IL-12/23p40 present in supernatants of DCs co-cultured with tachyzoites (as described in A ) was measured by ELISA. The data shown are representative of six experiments. C , CD11c + splenic DCs were incubated with YFP-expressing tachyzoites (2:1 ratio) for 20 h, and brefeldin A was added during the last 6 h of the culture. IL-12 production was analyzed by intracellular cytokine staining. As a positive control, DCs were stimulated with recombinant T. gondii profilin (1 μg/ml) as described above. D , mice were infected intraperitoneally with 10 5 YFP-expressing tachyzoites. Twenty hours later spleen was isolated and the percentage of IL-12 producing and infected cells (YFP + ) was analyzed by intracellular cytokine staining after incubation of splenocytes for 6 h in the presence of brefeldin A. E , splenocytes depleted of DCs ( Sp ) were placed in the bottom of a Transwell plate (separating membrane: 0.4-μm pores) and stimulated with live parental profilin-sufficient strain tachyzoites ( Toxo , ΔTgPRFe/TgPRFi strain), recombinant profilin (1 μg/ml), or profilin-deficient strain tachyzoites ( TgPRF KO Toxo , ΔTgPRFe/TgPRFi strain+ATc) in the presence or absence of DCs (upper portion of Transwell plate). As a positive control, DCs were cultivated with tachyzoites or recombinant profilin without a separating membrane. IL-12/23p40 present in supernatants was measured by ELISA. The data shown are representative of three performed experiments. Error bars , S.D.

    Article Snippet: LPS, cytochalasin D, chloroquine, brefeldin A, and bafilomycin were purchased from Sigma.

    Techniques: Infection, Purification, Incubation, Expressing, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Positive Control, Recombinant, Mouse Assay, Isolation

    Effect of endosomal inhibitors on the entry of  Mr Nvc VLPs into Sf9 cells. Sf9 cells were pre-incubated with different endosomal inhibitors: (A(iii)) cytochalasin D (2 µM), (A(iv)) NH 4 Cl (10 mM), (A(v)) CPZ (50 µM), (A(vi)) methyl- β -cyclodextrin (2 mM) and (A(vii)) genistein (100 µM).  Mr Nvc VLPs labelled with NHS-fluorescein (F- Mr Nvc VLPs; 25 mg/ml) were added to each pre-treated sample and incubated for 16 h in the presence of endosomal inhibitors. Bars, 20 µm. (A(ii)) Sf9 cells added with F- Mr Nvc VLPs but without any inhibitor serve as positive control, whereas (A(i)) Sf9 cells without any inhibitor nor F- Mr Nvc VLPs serve as negative control. (B) MTT assay showing the viability of Sf9 cells in the presence of inhibitors.

    Journal: PeerJ

    Article Title: Tracking the virus-like particles of Macrobrachium rosenbergii nodavirus in insect cells

    doi: 10.7717/peerj.2947

    Figure Lengend Snippet: Effect of endosomal inhibitors on the entry of Mr Nvc VLPs into Sf9 cells. Sf9 cells were pre-incubated with different endosomal inhibitors: (A(iii)) cytochalasin D (2 µM), (A(iv)) NH 4 Cl (10 mM), (A(v)) CPZ (50 µM), (A(vi)) methyl- β -cyclodextrin (2 mM) and (A(vii)) genistein (100 µM). Mr Nvc VLPs labelled with NHS-fluorescein (F- Mr Nvc VLPs; 25 mg/ml) were added to each pre-treated sample and incubated for 16 h in the presence of endosomal inhibitors. Bars, 20 µm. (A(ii)) Sf9 cells added with F- Mr Nvc VLPs but without any inhibitor serve as positive control, whereas (A(i)) Sf9 cells without any inhibitor nor F- Mr Nvc VLPs serve as negative control. (B) MTT assay showing the viability of Sf9 cells in the presence of inhibitors.

    Article Snippet: The Sf900II SFM medium was removed and the cells were pre-incubated in 2 ml medium for 1 h at RT with: cytochalasin D (2 µM; Calbiochem, CA, USA), NH4 Cl (10 mM; Bio Basic Inc., NY, USA), chlorpromazine (CPZ: 50 µM; Santa Cruz Biotechnology, Texas, USA), methyl-β -cyclodextrin (2 mM; Sigma-Aldrich, MO, USA) and genistein (100 µM; Calbiochem, CA, USA).

    Techniques: Incubation, Positive Control, Negative Control, MTT Assay