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  • 95
    Millipore nocodazole
    <t>Nocodazole</t> treatment affects IBDV VP3 subcellular distribution. pYFP-SialT2- and pYFP-GalNAcT-transfected and infected HeLa cells were treated with 2 mM nocodazole (ND) or left in control medium. At 36 h p.i., cells were washed and processed for indirect
    Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 12970 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Merck KGaA nocodazole
    <t>Nocodazole</t> treatment affects IBDV VP3 subcellular distribution. pYFP-SialT2- and pYFP-GalNAcT-transfected and infected HeLa cells were treated with 2 mM nocodazole (ND) or left in control medium. At 36 h p.i., cells were washed and processed for indirect
    Nocodazole, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 96/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore inhibitors nocodazole
    Interplay between septins and the actomyosin cytoskeleton in cell shrinkage. (a) Treatment with inhibitors of MyoII (blebbistatin), actin polymerization (latrunculin B), or microtubule polymerization <t>(nocodazole)</t> does not influence initial or maximum cell size in the flow cytometry osmotic swelling assay. AU, arbitrary unit. (b) Cortical retraction in this assay is slowed by blebbistatin or latrunculin but unaffected by nocodazole. (a and b) n = 7. (c) There is no additive or synergistic effect on cortical retraction of latrunculin B (Lat B) treatment of SEPT7KD cells. Lines indicate the groups between which statistical posttests were performed after analysis of variance. n = 5. (a–c) Error bars represent SD. (d and e) Illustration and quantification of anti-SEPT7 staining of wild-type cells showing that formation of septin filaments (white arrowheads) and rings (red arrowheads) is normal in cells treated with blebbistatin (pooled data from two independent experiments). Insets in d show septin rings in the boxed areas. Hypo, hypotonic; Iso, isotonic. (f) Imaging of SEPT6-GFP–expressing cells demonstrates septin aggregation into rings with latrunculin B treatment under isotonic conditions, whereas no such aggregation was observed with jasplakinolide (Jasp.) treatment. The inset shows latrunculin B–induced septin rings in greater detail. (g) Confocal imaging of cells expressing SEPT6-GFP and LifeAct-Ruby showing actin ruffles with subsequent recruitment of SEPT6-GFP to their bases. Bars, 10 µm.
    Inhibitors Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore graminearum nocodazole
    Interplay between septins and the actomyosin cytoskeleton in cell shrinkage. (a) Treatment with inhibitors of MyoII (blebbistatin), actin polymerization (latrunculin B), or microtubule polymerization <t>(nocodazole)</t> does not influence initial or maximum cell size in the flow cytometry osmotic swelling assay. AU, arbitrary unit. (b) Cortical retraction in this assay is slowed by blebbistatin or latrunculin but unaffected by nocodazole. (a and b) n = 7. (c) There is no additive or synergistic effect on cortical retraction of latrunculin B (Lat B) treatment of SEPT7KD cells. Lines indicate the groups between which statistical posttests were performed after analysis of variance. n = 5. (a–c) Error bars represent SD. (d and e) Illustration and quantification of anti-SEPT7 staining of wild-type cells showing that formation of septin filaments (white arrowheads) and rings (red arrowheads) is normal in cells treated with blebbistatin (pooled data from two independent experiments). Insets in d show septin rings in the boxed areas. Hypo, hypotonic; Iso, isotonic. (f) Imaging of SEPT6-GFP–expressing cells demonstrates septin aggregation into rings with latrunculin B treatment under isotonic conditions, whereas no such aggregation was observed with jasplakinolide (Jasp.) treatment. The inset shows latrunculin B–induced septin rings in greater detail. (g) Confocal imaging of cells expressing SEPT6-GFP and LifeAct-Ruby showing actin ruffles with subsequent recruitment of SEPT6-GFP to their bases. Bars, 10 µm.
    Graminearum Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore immunofluorescence nocodazole
    The myo2 mutant, which requires Smy1p at permissive temperature, does not need microtubules for bud growth. Wild-type (SLY248; triangles ) or myo2 -mutant (the average of counts using SLY250 and SLY251; circles ) cells growing exponentially in rich medium (YM-P) at 22° were treated at 0 h with 10 ( a–c ) or 15 ( d ) μg/ml <t>nocodazole,</t> and counts were made of unbudded ( b ), small-budded ( c ), and large-budded ( a and d ) cells. Results of two separate experiments are shown, indicated by open and closed symbols. At each time point, samples were fixed and counted or were stained for microtubules, as described in Fig. 1 . Addition of carrier DMSO alone had no effect on the proportions of unbudded versus budded cells (data not shown). In cells treated with 10 μg/ml nocodazole, all detectable cytoplasmic microtubules had disappeared by 30 min and started to reappear only at 6 3 / 4 h. (Spindles persisted for some time in 1–3% of the cells, and spindle pole bodies remained detectable in many cells throughout the experiment.) In cells treated with a lower (5 μg/ml, data not shown) or higher (15 μg/ml [ d ]) nocodazole dose, cytoplasmic microtubules reappeared at ∼3 3/4 h. In mock-treated cultures (DMSO alone), virtually all cells had detectable cytoplasmic microtubules at all times.
    Immunofluorescence Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore microtubule inhibitor nocodazole
    DY131 delays chromosome segregation in mitosis A. Individual frames representative of prophase, metaphase, and anaphase/telophase from live-cell confocal microscopy of MCF7 cells stably expressing GFP-H2B. Cells were accumulated in G2 by exposure to <t>nocodazole,</t> then released into DY131 or DMSO control. Arrows denote cells of interest. B. Quantitation of time elapsed from chromatin condensation (prophase) to anaphase in MCF7 cells stably expressing GFP-H2B after release from nocodazole block into DY131 or DMSO control. N = 4 – 11 cells, one-way ANOVA with Tukey's post-test.
    Microtubule Inhibitor Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore chromosome harvest nocodazole
    Chromosome length as a parameter for metaphase spread quality for karyotype analysis. The average length of chromosome 1 (here with the centromeric region marked in red) was measured using the image analysis package MetaMorph v7.6 (example above), and compared between the <t>nocodazole</t> 16 h/buffered hypotonic harvest and the demecolcine 16 h/buffered hypotonic harvest
    Chromosome Harvest Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore microtubule destabilizer nocodazole
    Chromosome length as a parameter for metaphase spread quality for karyotype analysis. The average length of chromosome 1 (here with the centromeric region marked in red) was measured using the image analysis package MetaMorph v7.6 (example above), and compared between the <t>nocodazole</t> 16 h/buffered hypotonic harvest and the demecolcine 16 h/buffered hypotonic harvest
    Microtubule Destabilizer Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore microtubule antagonists nocodazole
    Stabilization of microtubules at focal adhesions. (A–D ) Figure shows a 3T3 fibroblast that was fixed and triple labeled for actin ( D ), paxillin ( B ), and tubulin ( A and C ) after treatment with 1.5 μg/ml <t>nocodazole</t> for 10 min. All peripheral microtubules disassembled, except those whose ends targeted focal adhesions ( arrowheads ). ( E ) Video sequence showing the stabilization of a shrinking microtubule at a focal adhesion. Goldfish fibroblast co- injected with vinculin and tubulin. Frames are taken from a video sequence for which nocodazole (1.5 μg/ ml) was added at time 0. One of a pair of microtubules that extended to the periphery at the beginning of the sequence ( white arrowhead ) was prevented from shrinking beyond an adhesion site over which it passed ( arrow ). Eventually, it shrank into this adhesion site via depolymerization at its minus end ( black arrowhead ). Bars, 5 μm.
    Microtubule Antagonists Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore microtubular disrupting agent nocodazole
    Effect of BFA and <t>nocodazole</t> treatment on TGEV assembly. (A) Large virions (arrowheads) and small dense viral particles (arrows) coexist within the Golgi complex (G) of normal infected cells. (B) BFA causes the disappearance of the Golgi complex as a distinguishable structure, together with the formation of large cisternae (asterisks), some of them apparently derived from the rough endoplasmic reticulum (RER), since they have ribosomes (r) attached. TGEV virions assemble in association with these cisternae (arrowheads point to budding profiles). Large viral particles with an electron-dense internal periphery and clear center (arrows) accumulate in these conditions. Some ERGIC-like tubular membranes are visible in these cells (pairs of arrows). (C) Abnormal Golgi stack (G) from a nocodazole-treated infected ST cell. Both budding profiles (arrowheads) and small dense virions (arrows) are seen within the altered membranes of the stack. mi, mitochondria; pm, plasma membrane. Bar, 200 nm.
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    Millipore nocodazole washout assays nocodazole
    CENP-I increases the half-life of Mad1 at unattached kinetochores. (A) Images of Mad1-GFP FRAP in control, CENP-I–depleted, and ZM-treated cells arrested in <t>nocodazole.</t> (B) Recovery dynamics of Mad1-GFP after photobleaching demonstrating that CENP-I–depleted cells have a larger initial recovery of Mad1 and a faster turnover of stable Mad1. (C) Total recovery of Mad1-GFP at 120 s after photobleaching. (D) Scatter plot displaying the natural log of the normalized unrecovered fluorescence over time. The biphasic nature of Mad1 recovery is illustrated by overlaid lines. CENP-I–depleted cells have a fast phase of initial Mad1 recovery similar to controls but the pool of stable Mad1 in CENP-I–depleted cells has a greatly decreased half-life relative to control. Red arrows in A indicate FRAP targets. FRAP data are from n = 30 experiments. Error bars indicate standard deviation. *, P
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    Millipore benzimidazol 2 yl carbamate nocodazole
    CENP-I increases the half-life of Mad1 at unattached kinetochores. (A) Images of Mad1-GFP FRAP in control, CENP-I–depleted, and ZM-treated cells arrested in <t>nocodazole.</t> (B) Recovery dynamics of Mad1-GFP after photobleaching demonstrating that CENP-I–depleted cells have a larger initial recovery of Mad1 and a faster turnover of stable Mad1. (C) Total recovery of Mad1-GFP at 120 s after photobleaching. (D) Scatter plot displaying the natural log of the normalized unrecovered fluorescence over time. The biphasic nature of Mad1 recovery is illustrated by overlaid lines. CENP-I–depleted cells have a fast phase of initial Mad1 recovery similar to controls but the pool of stable Mad1 in CENP-I–depleted cells has a greatly decreased half-life relative to control. Red arrows in A indicate FRAP targets. FRAP data are from n = 30 experiments. Error bars indicate standard deviation. *, P
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    Millipore microtubule depolymerisation agent nocodazole
    CENP-I increases the half-life of Mad1 at unattached kinetochores. (A) Images of Mad1-GFP FRAP in control, CENP-I–depleted, and ZM-treated cells arrested in <t>nocodazole.</t> (B) Recovery dynamics of Mad1-GFP after photobleaching demonstrating that CENP-I–depleted cells have a larger initial recovery of Mad1 and a faster turnover of stable Mad1. (C) Total recovery of Mad1-GFP at 120 s after photobleaching. (D) Scatter plot displaying the natural log of the normalized unrecovered fluorescence over time. The biphasic nature of Mad1 recovery is illustrated by overlaid lines. CENP-I–depleted cells have a fast phase of initial Mad1 recovery similar to controls but the pool of stable Mad1 in CENP-I–depleted cells has a greatly decreased half-life relative to control. Red arrows in A indicate FRAP targets. FRAP data are from n = 30 experiments. Error bars indicate standard deviation. *, P
    Microtubule Depolymerisation Agent Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore microtubule depolymerizing drug nocodazole
    Cell and nuclear morphology under DMSO and <t>nocodazole</t> treatment. (A) Immunofluorescence images of untreated (this is same example as shown in A), DMSO control and nocodazole-treated MEF cells. Solid and open arrowheads indicate organized and disrupted
    Microtubule Depolymerizing Drug Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore microtubule disrupting drug nocodazole
    Formation of v-ErbA foci is microtubule-and dynein-dependent. (A) Treatment with nocodozole disrupts microtubules. HeLa cells were either left untreated, treated with DMSO (vehicle control), or treated with nocodozole (+NOC) for 20 h. Microtubules were visualized by immunostaining with Cy3-tagged anti-β-tubulin (red). Nuclei were stained for DNA with DAPI (blue) (B) Microtubule disruption prevents the formation of coalesced v-ErbA foci. HeLa cells were transfected with an expression vector for GFP-v-ErbA, and 16 h post-transfection were treated with <t>nocodazole</t> for 20 h. +NOC (diffuse), nocodazole-treated cell forming diffuse aggregates; +NOC (small aggregates), nocodazole-treated cell forming small aggregates, uniform in size; −NOC, untreated cell forming large juxtanuclear foci. (C) Bar graph summarizing the effect of nocodozole on v-ErbA foci size. Upon nocodazole treatment, there was a significant shift (p
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    Millipore microtubule depolymerizing agent nocodazole
    Colocalization of Tctex-1 and Rab3D with microtubules in osteoclasts and effect of <t>nocodazole</t> on Tctex-1–Rab3D interaction in vivo . Osteoclasts were briefly pre-extracted with microtubule stabilizing buffer, fixed in methanol, and then immunostained with the indicated antibodies. (A) A projected XY image stack of an osteoclast stained with anti-Tctex-1 and α-tubulin. (B) A single optical section of an osteoclast stained for anti-Rab3D and α-tubulin. Arrows depict Rab3D-bearing vesicles lying along individual microtubule filaments in magnified insets. (C) Treatment with the microtubule depolymerizing agent nocodazole (2 h) disrupts Rab3D–Tctex-1 distribution in osteoclasts. (D) Effect of brefeldin A, cytochalasin D (cyto D), and nocodazole on Tctex-1–Rab3D interaction as monitored by BRET (***, P
    Microtubule Depolymerizing Agent Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nocodazole emd calbiochem
    Colocalization of Tctex-1 and Rab3D with microtubules in osteoclasts and effect of <t>nocodazole</t> on Tctex-1–Rab3D interaction in vivo . Osteoclasts were briefly pre-extracted with microtubule stabilizing buffer, fixed in methanol, and then immunostained with the indicated antibodies. (A) A projected XY image stack of an osteoclast stained with anti-Tctex-1 and α-tubulin. (B) A single optical section of an osteoclast stained for anti-Rab3D and α-tubulin. Arrows depict Rab3D-bearing vesicles lying along individual microtubule filaments in magnified insets. (C) Treatment with the microtubule depolymerizing agent nocodazole (2 h) disrupts Rab3D–Tctex-1 distribution in osteoclasts. (D) Effect of brefeldin A, cytochalasin D (cyto D), and nocodazole on Tctex-1–Rab3D interaction as monitored by BRET (***, P
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    Millipore spindle poison nocodazole
    Colocalization of Tctex-1 and Rab3D with microtubules in osteoclasts and effect of <t>nocodazole</t> on Tctex-1–Rab3D interaction in vivo . Osteoclasts were briefly pre-extracted with microtubule stabilizing buffer, fixed in methanol, and then immunostained with the indicated antibodies. (A) A projected XY image stack of an osteoclast stained with anti-Tctex-1 and α-tubulin. (B) A single optical section of an osteoclast stained for anti-Rab3D and α-tubulin. Arrows depict Rab3D-bearing vesicles lying along individual microtubule filaments in magnified insets. (C) Treatment with the microtubule depolymerizing agent nocodazole (2 h) disrupts Rab3D–Tctex-1 distribution in osteoclasts. (D) Effect of brefeldin A, cytochalasin D (cyto D), and nocodazole on Tctex-1–Rab3D interaction as monitored by BRET (***, P
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    Merck KGaA nocodazole block
    Colocalization of Tctex-1 and Rab3D with microtubules in osteoclasts and effect of <t>nocodazole</t> on Tctex-1–Rab3D interaction in vivo . Osteoclasts were briefly pre-extracted with microtubule stabilizing buffer, fixed in methanol, and then immunostained with the indicated antibodies. (A) A projected XY image stack of an osteoclast stained with anti-Tctex-1 and α-tubulin. (B) A single optical section of an osteoclast stained for anti-Rab3D and α-tubulin. Arrows depict Rab3D-bearing vesicles lying along individual microtubule filaments in magnified insets. (C) Treatment with the microtubule depolymerizing agent nocodazole (2 h) disrupts Rab3D–Tctex-1 distribution in osteoclasts. (D) Effect of brefeldin A, cytochalasin D (cyto D), and nocodazole on Tctex-1–Rab3D interaction as monitored by BRET (***, P
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    Millipore microtubule polymerization inhibitor nocodazole
    Tri- and tetra-radial chromosomes and telomere fusion in U698 and JVM-2 cells. Cells were grown in the absence, or presence of 10 µM KU-55933 and 3 µM Olaparib for 48 h. <t>Nocodazole</t> was added for the last 6 h of incubation to increase the yield of metaphases. Metaphase spreads were prepared by standard cytogenetic methods. Tri- and tetra-radial chromosomes are indicated by filled arrows, telomere fusion is indicated by an asterix.
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    Millipore reversible microtubule depolymerizing drug nocodazole
    Bub3 depletion abrogates the spindle assembly checkpoint and causes chromosome misalignment. (A) Mitotic index of cells transfected with mock, Bub3, Bub1, or BubR1 siRNAs, before or after treatment with <t>nocodazole.</t> (B) Bub3-depleted cells exhibit abnormal
    Reversible Microtubule Depolymerizing Drug Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tubulin polymerization inhibitor nocodazole
    TUBA1B and TUBB3 are the interacting proteins of CLDN11; CLDN11 blocks cell migration by interfering with <t>tubulin</t> polymerization. a TW02 cells transfected with FLAG-tagged CLDN11 construct (C) or vector control plasmid (V) were harvested after 48 h. TW02 cell extracts and M2 beads were used for a co-immunoprecipitation assay. CLDN11-interacting proteins identified through LC–MS/MS and top-ranking proteins are listed in the table. b Immunoblot analyses were performed to confirm the interaction between FLAG-tagged CLDN11 and endogenous TUBA1B (left panel) or TUBB3 (right panel). c Subcellular distribution of exogenous FLAG-tagged CLDN11 and endogenous TUBA1B or TUBB3 in TW02 cells was assayed through immunofluorescence staining 24 h after transfection. The fluorescent signal (pixel) of each individual cell was quantitated by using the Zen 2.0 software ( n = 50). The proportion of colocalized fluorescent signals (%) is indicated (FLAG-tagged CLDN11, green; Tubulins, red; DAPI, blue). d Schematic illustrations of WT and four deletion FLAG-tagged CLDN11 molecules—transmembrane (TM), extracellular loop (ECL), intracellular loop (ICL), and C-terminus (C). Various FLAG-tagged CLDN11 deletion clones or a vector control were used to dissect the interacting domains on CLDN11 that are crucial for the interaction of endogenous TUBA1B and TUBB3. Input (3%) and immunoprecipitates (30% IP) were assayed through immunoblot analysis by using appropriate antibodies (anti-Flag, anti-TUBA1B and anti-TUBB3). The asterisks denote the major bands of ectopic CLDN11 in the immunoblot assays. e CLDN11 deletion clones were used to perform cell migration assay and to determine which domains on CLDN11 are necessary for blocking cell migration in TW02 cells. f Cell migration and ( g ) cell viability assays were performed in the presence or absence of <t>nocodazole</t> in TW02 or HK1 cells
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    TUBA1B and TUBB3 are the interacting proteins of CLDN11; CLDN11 blocks cell migration by interfering with <t>tubulin</t> polymerization. a TW02 cells transfected with FLAG-tagged CLDN11 construct (C) or vector control plasmid (V) were harvested after 48 h. TW02 cell extracts and M2 beads were used for a co-immunoprecipitation assay. CLDN11-interacting proteins identified through LC–MS/MS and top-ranking proteins are listed in the table. b Immunoblot analyses were performed to confirm the interaction between FLAG-tagged CLDN11 and endogenous TUBA1B (left panel) or TUBB3 (right panel). c Subcellular distribution of exogenous FLAG-tagged CLDN11 and endogenous TUBA1B or TUBB3 in TW02 cells was assayed through immunofluorescence staining 24 h after transfection. The fluorescent signal (pixel) of each individual cell was quantitated by using the Zen 2.0 software ( n = 50). The proportion of colocalized fluorescent signals (%) is indicated (FLAG-tagged CLDN11, green; Tubulins, red; DAPI, blue). d Schematic illustrations of WT and four deletion FLAG-tagged CLDN11 molecules—transmembrane (TM), extracellular loop (ECL), intracellular loop (ICL), and C-terminus (C). Various FLAG-tagged CLDN11 deletion clones or a vector control were used to dissect the interacting domains on CLDN11 that are crucial for the interaction of endogenous TUBA1B and TUBB3. Input (3%) and immunoprecipitates (30% IP) were assayed through immunoblot analysis by using appropriate antibodies (anti-Flag, anti-TUBA1B and anti-TUBB3). The asterisks denote the major bands of ectopic CLDN11 in the immunoblot assays. e CLDN11 deletion clones were used to perform cell migration assay and to determine which domains on CLDN11 are necessary for blocking cell migration in TW02 cells. f Cell migration and ( g ) cell viability assays were performed in the presence or absence of <t>nocodazole</t> in TW02 or HK1 cells
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    TUBA1B and TUBB3 are the interacting proteins of CLDN11; CLDN11 blocks cell migration by interfering with <t>tubulin</t> polymerization. a TW02 cells transfected with FLAG-tagged CLDN11 construct (C) or vector control plasmid (V) were harvested after 48 h. TW02 cell extracts and M2 beads were used for a co-immunoprecipitation assay. CLDN11-interacting proteins identified through LC–MS/MS and top-ranking proteins are listed in the table. b Immunoblot analyses were performed to confirm the interaction between FLAG-tagged CLDN11 and endogenous TUBA1B (left panel) or TUBB3 (right panel). c Subcellular distribution of exogenous FLAG-tagged CLDN11 and endogenous TUBA1B or TUBB3 in TW02 cells was assayed through immunofluorescence staining 24 h after transfection. The fluorescent signal (pixel) of each individual cell was quantitated by using the Zen 2.0 software ( n = 50). The proportion of colocalized fluorescent signals (%) is indicated (FLAG-tagged CLDN11, green; Tubulins, red; DAPI, blue). d Schematic illustrations of WT and four deletion FLAG-tagged CLDN11 molecules—transmembrane (TM), extracellular loop (ECL), intracellular loop (ICL), and C-terminus (C). Various FLAG-tagged CLDN11 deletion clones or a vector control were used to dissect the interacting domains on CLDN11 that are crucial for the interaction of endogenous TUBA1B and TUBB3. Input (3%) and immunoprecipitates (30% IP) were assayed through immunoblot analysis by using appropriate antibodies (anti-Flag, anti-TUBA1B and anti-TUBB3). The asterisks denote the major bands of ectopic CLDN11 in the immunoblot assays. e CLDN11 deletion clones were used to perform cell migration assay and to determine which domains on CLDN11 are necessary for blocking cell migration in TW02 cells. f Cell migration and ( g ) cell viability assays were performed in the presence or absence of <t>nocodazole</t> in TW02 or HK1 cells
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    TUBA1B and TUBB3 are the interacting proteins of CLDN11; CLDN11 blocks cell migration by interfering with <t>tubulin</t> polymerization. a TW02 cells transfected with FLAG-tagged CLDN11 construct (C) or vector control plasmid (V) were harvested after 48 h. TW02 cell extracts and M2 beads were used for a co-immunoprecipitation assay. CLDN11-interacting proteins identified through LC–MS/MS and top-ranking proteins are listed in the table. b Immunoblot analyses were performed to confirm the interaction between FLAG-tagged CLDN11 and endogenous TUBA1B (left panel) or TUBB3 (right panel). c Subcellular distribution of exogenous FLAG-tagged CLDN11 and endogenous TUBA1B or TUBB3 in TW02 cells was assayed through immunofluorescence staining 24 h after transfection. The fluorescent signal (pixel) of each individual cell was quantitated by using the Zen 2.0 software ( n = 50). The proportion of colocalized fluorescent signals (%) is indicated (FLAG-tagged CLDN11, green; Tubulins, red; DAPI, blue). d Schematic illustrations of WT and four deletion FLAG-tagged CLDN11 molecules—transmembrane (TM), extracellular loop (ECL), intracellular loop (ICL), and C-terminus (C). Various FLAG-tagged CLDN11 deletion clones or a vector control were used to dissect the interacting domains on CLDN11 that are crucial for the interaction of endogenous TUBA1B and TUBB3. Input (3%) and immunoprecipitates (30% IP) were assayed through immunoblot analysis by using appropriate antibodies (anti-Flag, anti-TUBA1B and anti-TUBB3). The asterisks denote the major bands of ectopic CLDN11 in the immunoblot assays. e CLDN11 deletion clones were used to perform cell migration assay and to determine which domains on CLDN11 are necessary for blocking cell migration in TW02 cells. f Cell migration and ( g ) cell viability assays were performed in the presence or absence of <t>nocodazole</t> in TW02 or HK1 cells
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    USP15 isoforms are dynamically expressed and differentially phosphorylated during the cell cycle. a , b A549 cells were synchronised using standard <t>thymidine/nocodazole</t> protocols to enrich for the indicated phases. a USP15 transcript levels remain stable during the cell cycle. Expression of USP15 splice variants and cyclin B1 (CCNB1) were quantified by qRT-PCR, mean data from three independent experiments are expressed relative to actin and normalised to the expression in asynchronous cells (As) for each splice variant. b USP15 protein expression levels oscillate through the cell cycle. USP15 was evaluated by immunoblotting; a representative gel is shown with quantification of total USP15 expression relative to actin below (mean of three independent experiments, error bars SD, one-way ANOVA with Tukey’s multiple comparison test * P ≤ 0.05, ** P ≤ 0.01). c – e USP15 isoform-1 increases in abundance by G2/M and becomes phosphorylated. A549 cells were depleted of USP15 isoforms as indicated and protein extracts were compared by immunoblotting for asynchronous cells (As) or cells arrested at G2/M. Separation by 4–12% gradient SDS-PAGE c showing quantification of the siCON2 samples d , and analysis by Phos-tag gel electrophoresis e
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    USP15 isoforms are dynamically expressed and differentially phosphorylated during the cell cycle. a , b A549 cells were synchronised using standard <t>thymidine/nocodazole</t> protocols to enrich for the indicated phases. a USP15 transcript levels remain stable during the cell cycle. Expression of USP15 splice variants and cyclin B1 (CCNB1) were quantified by qRT-PCR, mean data from three independent experiments are expressed relative to actin and normalised to the expression in asynchronous cells (As) for each splice variant. b USP15 protein expression levels oscillate through the cell cycle. USP15 was evaluated by immunoblotting; a representative gel is shown with quantification of total USP15 expression relative to actin below (mean of three independent experiments, error bars SD, one-way ANOVA with Tukey’s multiple comparison test * P ≤ 0.05, ** P ≤ 0.01). c – e USP15 isoform-1 increases in abundance by G2/M and becomes phosphorylated. A549 cells were depleted of USP15 isoforms as indicated and protein extracts were compared by immunoblotting for asynchronous cells (As) or cells arrested at G2/M. Separation by 4–12% gradient SDS-PAGE c showing quantification of the siCON2 samples d , and analysis by Phos-tag gel electrophoresis e
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    USP15 isoforms are dynamically expressed and differentially phosphorylated during the cell cycle. a , b A549 cells were synchronised using standard <t>thymidine/nocodazole</t> protocols to enrich for the indicated phases. a USP15 transcript levels remain stable during the cell cycle. Expression of USP15 splice variants and cyclin B1 (CCNB1) were quantified by qRT-PCR, mean data from three independent experiments are expressed relative to actin and normalised to the expression in asynchronous cells (As) for each splice variant. b USP15 protein expression levels oscillate through the cell cycle. USP15 was evaluated by immunoblotting; a representative gel is shown with quantification of total USP15 expression relative to actin below (mean of three independent experiments, error bars SD, one-way ANOVA with Tukey’s multiple comparison test * P ≤ 0.05, ** P ≤ 0.01). c – e USP15 isoform-1 increases in abundance by G2/M and becomes phosphorylated. A549 cells were depleted of USP15 isoforms as indicated and protein extracts were compared by immunoblotting for asynchronous cells (As) or cells arrested at G2/M. Separation by 4–12% gradient SDS-PAGE c showing quantification of the siCON2 samples d , and analysis by Phos-tag gel electrophoresis e
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    USP15 isoforms are dynamically expressed and differentially phosphorylated during the cell cycle. a , b A549 cells were synchronised using standard <t>thymidine/nocodazole</t> protocols to enrich for the indicated phases. a USP15 transcript levels remain stable during the cell cycle. Expression of USP15 splice variants and cyclin B1 (CCNB1) were quantified by qRT-PCR, mean data from three independent experiments are expressed relative to actin and normalised to the expression in asynchronous cells (As) for each splice variant. b USP15 protein expression levels oscillate through the cell cycle. USP15 was evaluated by immunoblotting; a representative gel is shown with quantification of total USP15 expression relative to actin below (mean of three independent experiments, error bars SD, one-way ANOVA with Tukey’s multiple comparison test * P ≤ 0.05, ** P ≤ 0.01). c – e USP15 isoform-1 increases in abundance by G2/M and becomes phosphorylated. A549 cells were depleted of USP15 isoforms as indicated and protein extracts were compared by immunoblotting for asynchronous cells (As) or cells arrested at G2/M. Separation by 4–12% gradient SDS-PAGE c showing quantification of the siCON2 samples d , and analysis by Phos-tag gel electrophoresis e
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    USP15 isoforms are dynamically expressed and differentially phosphorylated during the cell cycle. a , b A549 cells were synchronised using standard <t>thymidine/nocodazole</t> protocols to enrich for the indicated phases. a USP15 transcript levels remain stable during the cell cycle. Expression of USP15 splice variants and cyclin B1 (CCNB1) were quantified by qRT-PCR, mean data from three independent experiments are expressed relative to actin and normalised to the expression in asynchronous cells (As) for each splice variant. b USP15 protein expression levels oscillate through the cell cycle. USP15 was evaluated by immunoblotting; a representative gel is shown with quantification of total USP15 expression relative to actin below (mean of three independent experiments, error bars SD, one-way ANOVA with Tukey’s multiple comparison test * P ≤ 0.05, ** P ≤ 0.01). c – e USP15 isoform-1 increases in abundance by G2/M and becomes phosphorylated. A549 cells were depleted of USP15 isoforms as indicated and protein extracts were compared by immunoblotting for asynchronous cells (As) or cells arrested at G2/M. Separation by 4–12% gradient SDS-PAGE c showing quantification of the siCON2 samples d , and analysis by Phos-tag gel electrophoresis e
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    A, Microtubule disruption abrogates the inhibitory effect of cGMP on TGF-β-induced PAI-1 mRNA expression. Serum-starved PASMC were pretreated with <t>nocodazole</t> (5 μg/ml) or vehicle (control) for 1 h, followed by cGMP (0.5 m m ) for 1 h, and
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    Image Search Results


    Nocodazole treatment affects IBDV VP3 subcellular distribution. pYFP-SialT2- and pYFP-GalNAcT-transfected and infected HeLa cells were treated with 2 mM nocodazole (ND) or left in control medium. At 36 h p.i., cells were washed and processed for indirect

    Journal: Journal of Virology

    Article Title: The Endosomal Pathway and the Golgi Complex Are Involved in the Infectious Bursal Disease Virus Life Cycle

    doi: 10.1128/JVI.03152-12

    Figure Lengend Snippet: Nocodazole treatment affects IBDV VP3 subcellular distribution. pYFP-SialT2- and pYFP-GalNAcT-transfected and infected HeLa cells were treated with 2 mM nocodazole (ND) or left in control medium. At 36 h p.i., cells were washed and processed for indirect

    Article Snippet: Brefeldin A (BFA) and nocodazole (ND) were purchased from Sigma-Aldrich (Buenos Aires, Argentina) and were prepared according to the manufacturer's instructions.

    Techniques: Transfection, Infection

    (A) Cytoplasmic ER tubules oriented along the mother-bud axis are present through the neck in budded yeast cells. Haploid or diploid wild-type cells expressing Hmg1p-GFP were grown in YPD at 25°C to early log phase and examined by fluorescence microscopy. Arrowheads indicate two representative newly initiated buds that have not acquired detectable cortical ER tubules, whereas arrows point to expanding buds in which the structure of the ER tubules is similar to that in the mother cell. (B) Nocodazole treatment does not significantly affect the inheritance of cortical ER. SFNY1054 (top) and SFNY1055 (bottom) were incubated for 3 h at 25°C in YPD containing 15 μg/ml nocodazole. After the incubation, cells were fixed and stained with 25 ng/ml DAPI. Arrows point to the enlarged buds of cells blocked in nuclear division.

    Journal: Molecular Biology of the Cell

    Article Title: Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae

    doi:

    Figure Lengend Snippet: (A) Cytoplasmic ER tubules oriented along the mother-bud axis are present through the neck in budded yeast cells. Haploid or diploid wild-type cells expressing Hmg1p-GFP were grown in YPD at 25°C to early log phase and examined by fluorescence microscopy. Arrowheads indicate two representative newly initiated buds that have not acquired detectable cortical ER tubules, whereas arrows point to expanding buds in which the structure of the ER tubules is similar to that in the mother cell. (B) Nocodazole treatment does not significantly affect the inheritance of cortical ER. SFNY1054 (top) and SFNY1055 (bottom) were incubated for 3 h at 25°C in YPD containing 15 μg/ml nocodazole. After the incubation, cells were fixed and stained with 25 ng/ml DAPI. Arrows point to the enlarged buds of cells blocked in nuclear division.

    Article Snippet: When cells were treated with nocodazole, SFNY1054 and SFNY1055 were cultured in YPD to log phase, pelleted, and then resuspended in fresh YPD containing 15 μg/ml nocodazole (3.3 mg/ml stock in dimethyl sulfoxide; Sigma-Aldrich, St. Louis, MO) or 0.45% of dimethyl sulfoxide as a control.

    Techniques: Expressing, Fluorescence, Microscopy, Incubation, Staining

    Syntabulin and PICK1 comigrate along microtubule structures. ( A ) COS7 cells were transfected with GFP-Syntabulin and RFP-PICK1 and examined under a live-imaging microscope with both green- and red-fluorescent channels; images were collected for 2 min at 2-s intervals. The “snapshot” shows the colocalization of PICK1 and syntabulin at a single time point, and the Z-projection shows the movement trajectories of PICK1 and syntabulin over 2 min. The merged image shows that the trajectory of PICK1 overlapped with that of syntabulin, which was juxtaposed to syntabulin-induced microtubule bundles. ( B ) PICK1 and syntabulin vesicle movement at different time point in the cell presented in ( A ). Arrowheads indicate the position of a single PICK1-syntabulin-containing vesicle at 10-s intervals. The kymograph also shows the overlapped movement trace of PICK1 and syntabulin. ( C ) PICK1-syntabulin-containing vesicles move in a microtubule-dependent but not actin-dependent manner. The snapshots show the original position of PICK1 and syntabulin proteins before drug treatment, and the Z-projection shows the movement trajectories of both proteins before and after drug treatment. Noco, nocodazole; CytoD, cytochalasin D. Scale bar = 10 μm in all panels.

    Journal: Scientific Reports

    Article Title: Syntabulin regulates the trafficking of PICK1-containing vesicles in neurons

    doi: 10.1038/srep20924

    Figure Lengend Snippet: Syntabulin and PICK1 comigrate along microtubule structures. ( A ) COS7 cells were transfected with GFP-Syntabulin and RFP-PICK1 and examined under a live-imaging microscope with both green- and red-fluorescent channels; images were collected for 2 min at 2-s intervals. The “snapshot” shows the colocalization of PICK1 and syntabulin at a single time point, and the Z-projection shows the movement trajectories of PICK1 and syntabulin over 2 min. The merged image shows that the trajectory of PICK1 overlapped with that of syntabulin, which was juxtaposed to syntabulin-induced microtubule bundles. ( B ) PICK1 and syntabulin vesicle movement at different time point in the cell presented in ( A ). Arrowheads indicate the position of a single PICK1-syntabulin-containing vesicle at 10-s intervals. The kymograph also shows the overlapped movement trace of PICK1 and syntabulin. ( C ) PICK1-syntabulin-containing vesicles move in a microtubule-dependent but not actin-dependent manner. The snapshots show the original position of PICK1 and syntabulin proteins before drug treatment, and the Z-projection shows the movement trajectories of both proteins before and after drug treatment. Noco, nocodazole; CytoD, cytochalasin D. Scale bar = 10 μm in all panels.

    Article Snippet: HEK293T cells were transfected using the calcium phosphate coprecipitation method, and the medium was completely changed after 9 h. COS7 cells were transfected using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instruction and the medium was completely changed after 3 h. Nocodazole (Sigma, M1414) and cytochalasin D (Sigma, C8273) were prepared as 1000× stock solutions in DMSO and used at working concentrations of 5 and 2 μg/mL, respectively, for 20–40 min (as indicated).

    Techniques: Transfection, Imaging, Microscopy

    Localization of LL5β and CLASP1 after nocodazole and cytochalasin D treatment. (A) eGFP-LL5β localization after control DMSO (left), nocodazole (middle), and cytochalasin D treatment for 2 h. (B) Endogenous CLASP1 localization after control DMSO (left), nocodazole (middle), and cytochalasin D treatment for 1.5 h. Broken lines indicate the apical limit of the epiblast. Bars, 20 µm.

    Journal: The Journal of Cell Biology

    Article Title: Epiblast integrity requires CLASP and Dystroglycan-mediated microtubule anchoring to the basal cortex

    doi: 10.1083/jcb.201302075

    Figure Lengend Snippet: Localization of LL5β and CLASP1 after nocodazole and cytochalasin D treatment. (A) eGFP-LL5β localization after control DMSO (left), nocodazole (middle), and cytochalasin D treatment for 2 h. (B) Endogenous CLASP1 localization after control DMSO (left), nocodazole (middle), and cytochalasin D treatment for 1.5 h. Broken lines indicate the apical limit of the epiblast. Bars, 20 µm.

    Article Snippet: For chemical treatment, embryos were grown in standard New culture setting and treated with Nocodazole (T7402; Sigma-Aldrich) or Cytochalasin D (C8273; Sigma-Aldrich) dissolved in thin albumen at a final concentration of 5 µg/ml.

    Techniques:

    Cav1-containing structures move long distances in activated cells. (A) Large peripheral areas of CV-1 cells expressing Cav1-GFP were bleached (marked areas in bleach panels), and the movement of Cav1-GFP into the bleached area was monitored omitting the 5-μm region closest to the bleach boundary (marked in 15 min panels). The experiment was performed in the absence (untreated, upper panels) and the presence of SV40 (1 h incubation, MOI 60; +SV40, lower panels). Before (Prebleach), immediately after (Bleach), and 15 min after (15 min) bleaching are shown. Note the increase in long-distance movement in the presence of SV40 (see Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1 ). Bars, 10 μm. (B) Recovery of fluorescence due to the long-distance movement of Cav1-GFP in CV-1 cells increases after addition of SV40, vanadate, or latA. The CV-1 cells expressing Cav1-GFP were either untreated, exposed to SV40 (MOI 60) for 1 h, to 1 mM vanadate for 1 h, to 5 μM nocodazole for 30 min, to 5 μM nocodazole for 30 min and then 1 h to SV40 (MOI 60), or to 0.8 μM latA for 10 min before the FRAP experiments. The fluorescence recovery was quantified in the bleached area omitting the 5 μm region closest to the bleach boundary (marked in 15 min panels in A) after 15 min. Recovery was calculated by measuring the fluorescence intensity in the defined area before and 15 min after bleaching (see Videos 5–7). The error bars indicate standard deviations of five independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Assembly and trafficking of caveolar domains in the cell

    doi: 10.1083/jcb.200506103

    Figure Lengend Snippet: Cav1-containing structures move long distances in activated cells. (A) Large peripheral areas of CV-1 cells expressing Cav1-GFP were bleached (marked areas in bleach panels), and the movement of Cav1-GFP into the bleached area was monitored omitting the 5-μm region closest to the bleach boundary (marked in 15 min panels). The experiment was performed in the absence (untreated, upper panels) and the presence of SV40 (1 h incubation, MOI 60; +SV40, lower panels). Before (Prebleach), immediately after (Bleach), and 15 min after (15 min) bleaching are shown. Note the increase in long-distance movement in the presence of SV40 (see Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1 ). Bars, 10 μm. (B) Recovery of fluorescence due to the long-distance movement of Cav1-GFP in CV-1 cells increases after addition of SV40, vanadate, or latA. The CV-1 cells expressing Cav1-GFP were either untreated, exposed to SV40 (MOI 60) for 1 h, to 1 mM vanadate for 1 h, to 5 μM nocodazole for 30 min, to 5 μM nocodazole for 30 min and then 1 h to SV40 (MOI 60), or to 0.8 μM latA for 10 min before the FRAP experiments. The fluorescence recovery was quantified in the bleached area omitting the 5 μm region closest to the bleach boundary (marked in 15 min panels in A) after 15 min. Recovery was calculated by measuring the fluorescence intensity in the defined area before and 15 min after bleaching (see Videos 5–7). The error bars indicate standard deviations of five independent experiments.

    Article Snippet: To see the effect of ligand and drugs, the cells were treated as follows: infected with SV40 (MOI 60) for 1 h, treated with 1 mM sodium orthovanadate (vanadate; Calbiochem-Novabiochem) for 1 h, 100 μM genistein (Sigma-Aldrich) for 30 min, and then incubated further 1 h with SV40 (MOI 60), treated with 5 μM nocodazole (Sigma-Aldrich) for 30 min, treated with 5 μM nocodazole for 30 min, and then incubated with SV40 (MOI 60) for 1 h, or treated with 0.8 μM latA (Molecular Probes) for 10 min. A defined region was bleached at full laser power (100% power, 100% transmission, 25 iterations) using the 488 nm line from a 30 mW Argon/2 laser.

    Techniques: Expressing, Incubation, Fluorescence

    Assay of microtubule turnover in permeabilized NIH3T3 cells reconstituted with mammalian interphase cell extracts. Permeabilized NIH 3T3 cells were incubated at 34°C with TTL − cell extracts containing 1 μM purified Glu-tubulin, ATP-regenerating system, and 5 μM okadaic acid. ( A–B ) Double immunostaining of interphase microtubule arrays in permeabilized cells with Tyr-tubulin monoclonal antibody YL1/2 ( green ) and Glu-tubulin antibody ( red ) after a 30-min incubation with cell extracts. ( A ) The tubulin from the extract forms short tails on preexisting interphase microtubules, but fails to invade the interphase network. ( B ) 2× enlargement of a peripheral zone of the cell showing the Glu-tubulin tails at the ends of Tyr-microtubules. ( C ) Immunostaining of interphase permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cell extracts were supplemented with 20 μM nocodazole; this control experiment shows that microtubules were nocodazole-sensitive during microtubule turnover assay. Bars: ( A and C ) 10 μm; ( B ) 5 μm. ( D ) Quantitative analysis of microtubule turnover. Cells were incubated with TTL − cell extracts as described in A . At the indicated time points, cells were fixed and stained with Tyr-tubulin antibody, and the amount of Tyr-tubulin in interphase microtubule networks was quantified as described in Materials and Methods.

    Journal: The Journal of Cell Biology

    Article Title: Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

    doi:

    Figure Lengend Snippet: Assay of microtubule turnover in permeabilized NIH3T3 cells reconstituted with mammalian interphase cell extracts. Permeabilized NIH 3T3 cells were incubated at 34°C with TTL − cell extracts containing 1 μM purified Glu-tubulin, ATP-regenerating system, and 5 μM okadaic acid. ( A–B ) Double immunostaining of interphase microtubule arrays in permeabilized cells with Tyr-tubulin monoclonal antibody YL1/2 ( green ) and Glu-tubulin antibody ( red ) after a 30-min incubation with cell extracts. ( A ) The tubulin from the extract forms short tails on preexisting interphase microtubules, but fails to invade the interphase network. ( B ) 2× enlargement of a peripheral zone of the cell showing the Glu-tubulin tails at the ends of Tyr-microtubules. ( C ) Immunostaining of interphase permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cell extracts were supplemented with 20 μM nocodazole; this control experiment shows that microtubules were nocodazole-sensitive during microtubule turnover assay. Bars: ( A and C ) 10 μm; ( B ) 5 μm. ( D ) Quantitative analysis of microtubule turnover. Cells were incubated with TTL − cell extracts as described in A . At the indicated time points, cells were fixed and stained with Tyr-tubulin antibody, and the amount of Tyr-tubulin in interphase microtubule networks was quantified as described in Materials and Methods.

    Article Snippet: Materials Nocodazole was purchased from Sigma Aldrich (Strasbourg, France), and colchicine was obtained from Merck (Darmstadt, Germany).

    Techniques: Incubation, Purification, Double Immunostaining, Immunostaining, Turnover Assay, Staining

    Analysis of the mechanism of nocodazole action. ( A–C ) Immunostaining of interphasic permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cells were permeabilized and then incubated for 30 min at 34°C with NIH3T3 cells extracts supplemented with 5 μM pure tubulin in the presence of: ( A ) ATP-regenerating system and 5 μM okadaic acid; ( B ) ATP-regenerating system, 5 μM okadaic acid and 20 μM nocodazole; and ( C ) ATP-regenerating system, 5 μM okadaic acid, 20 μM nocodazole, and 20 μM tubulin. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

    doi:

    Figure Lengend Snippet: Analysis of the mechanism of nocodazole action. ( A–C ) Immunostaining of interphasic permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cells were permeabilized and then incubated for 30 min at 34°C with NIH3T3 cells extracts supplemented with 5 μM pure tubulin in the presence of: ( A ) ATP-regenerating system and 5 μM okadaic acid; ( B ) ATP-regenerating system, 5 μM okadaic acid and 20 μM nocodazole; and ( C ) ATP-regenerating system, 5 μM okadaic acid, 20 μM nocodazole, and 20 μM tubulin. Bar, 10 μm.

    Article Snippet: Materials Nocodazole was purchased from Sigma Aldrich (Strasbourg, France), and colchicine was obtained from Merck (Darmstadt, Germany).

    Techniques: Immunostaining, Incubation

    Inhibition of nocodazole action in the presence of added CD complex. ( A–B ) Immunostaining of interphasic permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cells were permeabilized and then incubated for 30 min at 34°C with NIH3T3 cell extracts supplemented with 5 μM pure tubulin, ATP-regenerating system and 5 μM okadaic acid in the absence of CD complex ( A ), or in the presence of 5 μM CD complex ( B ). ( C–D ) Immunofluorescence analysis of the microtubule content of control ( C ) or CD complex–supplemented ( D ) cell extracts. Cell extracts supplemented with 10 μM tubulin and ATP-regenerating system were incubated for 30 min at 30°C. Aliquots were then incubated for 30 min in the absence ( C ) or presence of 5 μM CD complex ( D ). Microtubules were then cross-linked, spun onto coverslips, and immunostained as described in Saoudi et al. (1995) . ( E–F ) Immunostaining of interphase-permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cells were permeabilized and then incubated for 30 min at 34°C with NIH3T3 cell extracts supplemented with 5 μM pure tubulin, ATP-regenerating system, 5 μM okadaic acid, and 20 μM nocodazole in the absence of CD complex ( E ) or the presence of 5 μM CD complex ( F ). Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

    doi:

    Figure Lengend Snippet: Inhibition of nocodazole action in the presence of added CD complex. ( A–B ) Immunostaining of interphasic permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cells were permeabilized and then incubated for 30 min at 34°C with NIH3T3 cell extracts supplemented with 5 μM pure tubulin, ATP-regenerating system and 5 μM okadaic acid in the absence of CD complex ( A ), or in the presence of 5 μM CD complex ( B ). ( C–D ) Immunofluorescence analysis of the microtubule content of control ( C ) or CD complex–supplemented ( D ) cell extracts. Cell extracts supplemented with 10 μM tubulin and ATP-regenerating system were incubated for 30 min at 30°C. Aliquots were then incubated for 30 min in the absence ( C ) or presence of 5 μM CD complex ( D ). Microtubules were then cross-linked, spun onto coverslips, and immunostained as described in Saoudi et al. (1995) . ( E–F ) Immunostaining of interphase-permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cells were permeabilized and then incubated for 30 min at 34°C with NIH3T3 cell extracts supplemented with 5 μM pure tubulin, ATP-regenerating system, 5 μM okadaic acid, and 20 μM nocodazole in the absence of CD complex ( E ) or the presence of 5 μM CD complex ( F ). Bar, 10 μm.

    Article Snippet: Materials Nocodazole was purchased from Sigma Aldrich (Strasbourg, France), and colchicine was obtained from Merck (Darmstadt, Germany).

    Techniques: Inhibition, Immunostaining, Incubation, Immunofluorescence

    Reconstitution of microtubule sensitivity to nocodazole action in permeabilized NIH 3T3 cells. ( A–F ) Immunostaining of interphasic permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cells were permeabilized and then incubated for 30 min at 34°C with NIH3T3 cell extracts supplemented with 5 μM pure tubulin in the presence of: ( A ) no addition; ( B ) 20 μM nocodazole ( Noc ); ( C ) ATP-regenerating system; ( D ) ATP-regenerating system and 20 μM nocodazole; ( E ) ATP-regenerating system and 5 μM okadaic acid; and ( F ) ATP-regenerating system, 5 μM okadaic acid, and 20 μM nocodazole. Arrows show microtubule extensions arising from the polymerization of cell extract tubulin at the ends of interphase microtubules. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

    doi:

    Figure Lengend Snippet: Reconstitution of microtubule sensitivity to nocodazole action in permeabilized NIH 3T3 cells. ( A–F ) Immunostaining of interphasic permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cells were permeabilized and then incubated for 30 min at 34°C with NIH3T3 cell extracts supplemented with 5 μM pure tubulin in the presence of: ( A ) no addition; ( B ) 20 μM nocodazole ( Noc ); ( C ) ATP-regenerating system; ( D ) ATP-regenerating system and 20 μM nocodazole; ( E ) ATP-regenerating system and 5 μM okadaic acid; and ( F ) ATP-regenerating system, 5 μM okadaic acid, and 20 μM nocodazole. Arrows show microtubule extensions arising from the polymerization of cell extract tubulin at the ends of interphase microtubules. Bar, 10 μm.

    Article Snippet: Materials Nocodazole was purchased from Sigma Aldrich (Strasbourg, France), and colchicine was obtained from Merck (Darmstadt, Germany).

    Techniques: Immunostaining, Incubation

    Mapping RMI1 mitotic phosphorylation sites. ( A ) Schematic representation of full-length (FL) Xpress-RMI1 and truncated fragments; ( B ) 293T cells were transfected with the constructs shown above. Thirty-two hours later, cells were treated with nocodazole or not for 16 h prior to harvest. Extracted proteins were treated with λ-phosphatase or not as shown, then resolved by 8% polyacrylamide gel and blotted with anti-Xpress antibody.

    Journal: International Journal of Molecular Sciences

    Article Title: Accumulation and Phosphorylation of RecQ-Mediated Genome Instability Protein 1 (RMI1) at Serine 284 and Serine 292 during Mitosis

    doi: 10.3390/ijms161125965

    Figure Lengend Snippet: Mapping RMI1 mitotic phosphorylation sites. ( A ) Schematic representation of full-length (FL) Xpress-RMI1 and truncated fragments; ( B ) 293T cells were transfected with the constructs shown above. Thirty-two hours later, cells were treated with nocodazole or not for 16 h prior to harvest. Extracted proteins were treated with λ-phosphatase or not as shown, then resolved by 8% polyacrylamide gel and blotted with anti-Xpress antibody.

    Article Snippet: Chemicals Nocodazole (Sigma, St. Louis, MO, USA) was resuspended in DMSO to stock concentrations of 1 mg/mL and used at the dilution of 1:2000.

    Techniques: Transfection, Construct

    RMI1 accumulates and is phosphorylated in mitosis. ( A ) Mitotic cells were harvested by mechanical shake-off from the nocodazole-arrested HeLa cells and untreated asynchronous HeLa cells. Attached cells were considered as interphase cells. Upper panel: Total cell lysates were analyzed by Western blot; Lower panel: The relative RMI1 protein levels were calculated according to their densitometry readings, which were normalized by the corresponding Actin bands; ( B ) Nocodazole-treated cell lysates were subjected to the λ-phosphatase treatment or not. SO: shake-off cells; AT: attached cells. Numbers under the panels stand for the lane number.

    Journal: International Journal of Molecular Sciences

    Article Title: Accumulation and Phosphorylation of RecQ-Mediated Genome Instability Protein 1 (RMI1) at Serine 284 and Serine 292 during Mitosis

    doi: 10.3390/ijms161125965

    Figure Lengend Snippet: RMI1 accumulates and is phosphorylated in mitosis. ( A ) Mitotic cells were harvested by mechanical shake-off from the nocodazole-arrested HeLa cells and untreated asynchronous HeLa cells. Attached cells were considered as interphase cells. Upper panel: Total cell lysates were analyzed by Western blot; Lower panel: The relative RMI1 protein levels were calculated according to their densitometry readings, which were normalized by the corresponding Actin bands; ( B ) Nocodazole-treated cell lysates were subjected to the λ-phosphatase treatment or not. SO: shake-off cells; AT: attached cells. Numbers under the panels stand for the lane number.

    Article Snippet: Chemicals Nocodazole (Sigma, St. Louis, MO, USA) was resuspended in DMSO to stock concentrations of 1 mg/mL and used at the dilution of 1:2000.

    Techniques: Western Blot

    Ser284 and Ser292 are RMI1 mitotic phosphorylation sites. The indicated RMI1 mutants were transiently transfected into 293T cells followed by nocodazole treatment or not for 16 h. Ectopic expression of wild type (WT), T270V, S284A or S292A ( A ); or S284A/S292A, T270V mutation of RMI1 (238–625) fragment ( B ); ( C ) Ectopic expression of full-length wild-type RMI1 or S284A/S292A mutant. Nocodazole treatment and phosphatase treatment were applied as indicated. Quantified relative RMI1 protein levels are shown under the corresponding blots.

    Journal: International Journal of Molecular Sciences

    Article Title: Accumulation and Phosphorylation of RecQ-Mediated Genome Instability Protein 1 (RMI1) at Serine 284 and Serine 292 during Mitosis

    doi: 10.3390/ijms161125965

    Figure Lengend Snippet: Ser284 and Ser292 are RMI1 mitotic phosphorylation sites. The indicated RMI1 mutants were transiently transfected into 293T cells followed by nocodazole treatment or not for 16 h. Ectopic expression of wild type (WT), T270V, S284A or S292A ( A ); or S284A/S292A, T270V mutation of RMI1 (238–625) fragment ( B ); ( C ) Ectopic expression of full-length wild-type RMI1 or S284A/S292A mutant. Nocodazole treatment and phosphatase treatment were applied as indicated. Quantified relative RMI1 protein levels are shown under the corresponding blots.

    Article Snippet: Chemicals Nocodazole (Sigma, St. Louis, MO, USA) was resuspended in DMSO to stock concentrations of 1 mg/mL and used at the dilution of 1:2000.

    Techniques: Transfection, Expressing, Mutagenesis

    Mitotic RMI1 phosphorylation is partly reversed after roscovitine treatment. HeLa cells were left untreated or treated with nocodazole for 18 h. Thereafter, caffeine ( A ) or roscovitine ( B ) was added for 2 h. Cell lysates were analyzed by Western blotting.

    Journal: International Journal of Molecular Sciences

    Article Title: Accumulation and Phosphorylation of RecQ-Mediated Genome Instability Protein 1 (RMI1) at Serine 284 and Serine 292 during Mitosis

    doi: 10.3390/ijms161125965

    Figure Lengend Snippet: Mitotic RMI1 phosphorylation is partly reversed after roscovitine treatment. HeLa cells were left untreated or treated with nocodazole for 18 h. Thereafter, caffeine ( A ) or roscovitine ( B ) was added for 2 h. Cell lysates were analyzed by Western blotting.

    Article Snippet: Chemicals Nocodazole (Sigma, St. Louis, MO, USA) was resuspended in DMSO to stock concentrations of 1 mg/mL and used at the dilution of 1:2000.

    Techniques: Western Blot

    Phosphorylation of RMI1 at Ser284 and Ser292 does not disrupt the interaction between RMI1, TopoIIIα and BLM. 293T cells were transfected with FLAG-tagged Topo III α plus Xpress-tagged WT or phospho-mutant (S284A/S292A) RMI1, or GFP expression construct to monitor transfection efficiency, and serve as a negative control (all “−”). Thirty-two hours after transfection, the cells were subjected to nocodazole treatment for 16 h or not as indicated. ( A ) Whole cell extracts were incubated with FLAG-affinity gel. The precipitates were immunoblotted with anti-RMI1, anti-BLM and anti-FLAG antibodies. * Non-specific bands; ( B ) 40 μg of whole cell lysates were analyzed by western blot indicating the input proteins in IP.

    Journal: International Journal of Molecular Sciences

    Article Title: Accumulation and Phosphorylation of RecQ-Mediated Genome Instability Protein 1 (RMI1) at Serine 284 and Serine 292 during Mitosis

    doi: 10.3390/ijms161125965

    Figure Lengend Snippet: Phosphorylation of RMI1 at Ser284 and Ser292 does not disrupt the interaction between RMI1, TopoIIIα and BLM. 293T cells were transfected with FLAG-tagged Topo III α plus Xpress-tagged WT or phospho-mutant (S284A/S292A) RMI1, or GFP expression construct to monitor transfection efficiency, and serve as a negative control (all “−”). Thirty-two hours after transfection, the cells were subjected to nocodazole treatment for 16 h or not as indicated. ( A ) Whole cell extracts were incubated with FLAG-affinity gel. The precipitates were immunoblotted with anti-RMI1, anti-BLM and anti-FLAG antibodies. * Non-specific bands; ( B ) 40 μg of whole cell lysates were analyzed by western blot indicating the input proteins in IP.

    Article Snippet: Chemicals Nocodazole (Sigma, St. Louis, MO, USA) was resuspended in DMSO to stock concentrations of 1 mg/mL and used at the dilution of 1:2000.

    Techniques: Transfection, Mutagenesis, Expressing, Construct, Negative Control, Incubation, Western Blot

    Interplay between septins and the actomyosin cytoskeleton in cell shrinkage. (a) Treatment with inhibitors of MyoII (blebbistatin), actin polymerization (latrunculin B), or microtubule polymerization (nocodazole) does not influence initial or maximum cell size in the flow cytometry osmotic swelling assay. AU, arbitrary unit. (b) Cortical retraction in this assay is slowed by blebbistatin or latrunculin but unaffected by nocodazole. (a and b) n = 7. (c) There is no additive or synergistic effect on cortical retraction of latrunculin B (Lat B) treatment of SEPT7KD cells. Lines indicate the groups between which statistical posttests were performed after analysis of variance. n = 5. (a–c) Error bars represent SD. (d and e) Illustration and quantification of anti-SEPT7 staining of wild-type cells showing that formation of septin filaments (white arrowheads) and rings (red arrowheads) is normal in cells treated with blebbistatin (pooled data from two independent experiments). Insets in d show septin rings in the boxed areas. Hypo, hypotonic; Iso, isotonic. (f) Imaging of SEPT6-GFP–expressing cells demonstrates septin aggregation into rings with latrunculin B treatment under isotonic conditions, whereas no such aggregation was observed with jasplakinolide (Jasp.) treatment. The inset shows latrunculin B–induced septin rings in greater detail. (g) Confocal imaging of cells expressing SEPT6-GFP and LifeAct-Ruby showing actin ruffles with subsequent recruitment of SEPT6-GFP to their bases. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: The septin cytoskeleton facilitates membrane retraction during motility and blebbing

    doi: 10.1083/jcb.201105127

    Figure Lengend Snippet: Interplay between septins and the actomyosin cytoskeleton in cell shrinkage. (a) Treatment with inhibitors of MyoII (blebbistatin), actin polymerization (latrunculin B), or microtubule polymerization (nocodazole) does not influence initial or maximum cell size in the flow cytometry osmotic swelling assay. AU, arbitrary unit. (b) Cortical retraction in this assay is slowed by blebbistatin or latrunculin but unaffected by nocodazole. (a and b) n = 7. (c) There is no additive or synergistic effect on cortical retraction of latrunculin B (Lat B) treatment of SEPT7KD cells. Lines indicate the groups between which statistical posttests were performed after analysis of variance. n = 5. (a–c) Error bars represent SD. (d and e) Illustration and quantification of anti-SEPT7 staining of wild-type cells showing that formation of septin filaments (white arrowheads) and rings (red arrowheads) is normal in cells treated with blebbistatin (pooled data from two independent experiments). Insets in d show septin rings in the boxed areas. Hypo, hypotonic; Iso, isotonic. (f) Imaging of SEPT6-GFP–expressing cells demonstrates septin aggregation into rings with latrunculin B treatment under isotonic conditions, whereas no such aggregation was observed with jasplakinolide (Jasp.) treatment. The inset shows latrunculin B–induced septin rings in greater detail. (g) Confocal imaging of cells expressing SEPT6-GFP and LifeAct-Ruby showing actin ruffles with subsequent recruitment of SEPT6-GFP to their bases. Bars, 10 µm.

    Article Snippet: Inhibitors Nocodazole (Sigma-Aldrich) was used at 5 µM for flow cytometry experiments and 13 µM for cell cycle synchronization.

    Techniques: Flow Cytometry, Cytometry, Staining, Imaging, Expressing

    Septins regulate cortical stability. (a) D10 T cells crawling on ICAM-1–coated glass display periodic membrane blebs and protrusions (black arrowheads). (b) Fluorescent images of D10 T cells expressing GPI-mCherry and SEPT6-GFP indicate that leading edge protrusions (white arrowheads) retract into the septin collar. n = 18. (c) Control and SEPT7KD cells were cell cycle synchronized with nocodazole and then released for imaging. Time-lapse images indicate profound blebbing during cytokinesis among SEPT7KD cells. A blebbing index consisting of the number of blebs divided by the time observed times the cell perimeter was calculated for each cell and indicates significantly elevated blebbing in SEPT7KD cells. Error bars represent SEM. AU, arbitrary unit. (d) Differential interference contrast (DIC) and SEPT6-GFP fluorescence time-lapse images of cells undergoing mitosis as in c, demonstrating accumulations of septin complexes at the midbody late in cytokinesis. (e) A confocal image and linescan demonstrating that a population of SEPT6-GFP remains localized to the cortex during mitosis. The red line indicates the path of the linescan. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: The septin cytoskeleton facilitates membrane retraction during motility and blebbing

    doi: 10.1083/jcb.201105127

    Figure Lengend Snippet: Septins regulate cortical stability. (a) D10 T cells crawling on ICAM-1–coated glass display periodic membrane blebs and protrusions (black arrowheads). (b) Fluorescent images of D10 T cells expressing GPI-mCherry and SEPT6-GFP indicate that leading edge protrusions (white arrowheads) retract into the septin collar. n = 18. (c) Control and SEPT7KD cells were cell cycle synchronized with nocodazole and then released for imaging. Time-lapse images indicate profound blebbing during cytokinesis among SEPT7KD cells. A blebbing index consisting of the number of blebs divided by the time observed times the cell perimeter was calculated for each cell and indicates significantly elevated blebbing in SEPT7KD cells. Error bars represent SEM. AU, arbitrary unit. (d) Differential interference contrast (DIC) and SEPT6-GFP fluorescence time-lapse images of cells undergoing mitosis as in c, demonstrating accumulations of septin complexes at the midbody late in cytokinesis. (e) A confocal image and linescan demonstrating that a population of SEPT6-GFP remains localized to the cortex during mitosis. The red line indicates the path of the linescan. Bars, 10 µm.

    Article Snippet: Inhibitors Nocodazole (Sigma-Aldrich) was used at 5 µM for flow cytometry experiments and 13 µM for cell cycle synchronization.

    Techniques: Expressing, Imaging, Fluorescence

    The myo2 mutant, which requires Smy1p at permissive temperature, does not need microtubules for bud growth. Wild-type (SLY248; triangles ) or myo2 -mutant (the average of counts using SLY250 and SLY251; circles ) cells growing exponentially in rich medium (YM-P) at 22° were treated at 0 h with 10 ( a–c ) or 15 ( d ) μg/ml nocodazole, and counts were made of unbudded ( b ), small-budded ( c ), and large-budded ( a and d ) cells. Results of two separate experiments are shown, indicated by open and closed symbols. At each time point, samples were fixed and counted or were stained for microtubules, as described in Fig. 1 . Addition of carrier DMSO alone had no effect on the proportions of unbudded versus budded cells (data not shown). In cells treated with 10 μg/ml nocodazole, all detectable cytoplasmic microtubules had disappeared by 30 min and started to reappear only at 6 3 / 4 h. (Spindles persisted for some time in 1–3% of the cells, and spindle pole bodies remained detectable in many cells throughout the experiment.) In cells treated with a lower (5 μg/ml, data not shown) or higher (15 μg/ml [ d ]) nocodazole dose, cytoplasmic microtubules reappeared at ∼3 3/4 h. In mock-treated cultures (DMSO alone), virtually all cells had detectable cytoplasmic microtubules at all times.

    Journal: The Journal of Cell Biology

    Article Title: Smy1p, a Kinesin-related Protein That Does Not Require Microtubules

    doi:

    Figure Lengend Snippet: The myo2 mutant, which requires Smy1p at permissive temperature, does not need microtubules for bud growth. Wild-type (SLY248; triangles ) or myo2 -mutant (the average of counts using SLY250 and SLY251; circles ) cells growing exponentially in rich medium (YM-P) at 22° were treated at 0 h with 10 ( a–c ) or 15 ( d ) μg/ml nocodazole, and counts were made of unbudded ( b ), small-budded ( c ), and large-budded ( a and d ) cells. Results of two separate experiments are shown, indicated by open and closed symbols. At each time point, samples were fixed and counted or were stained for microtubules, as described in Fig. 1 . Addition of carrier DMSO alone had no effect on the proportions of unbudded versus budded cells (data not shown). In cells treated with 10 μg/ml nocodazole, all detectable cytoplasmic microtubules had disappeared by 30 min and started to reappear only at 6 3 / 4 h. (Spindles persisted for some time in 1–3% of the cells, and spindle pole bodies remained detectable in many cells throughout the experiment.) In cells treated with a lower (5 μg/ml, data not shown) or higher (15 μg/ml [ d ]) nocodazole dose, cytoplasmic microtubules reappeared at ∼3 3/4 h. In mock-treated cultures (DMSO alone), virtually all cells had detectable cytoplasmic microtubules at all times.

    Article Snippet: Nocodazole Treatment and Immunofluorescence Nocodazole ( Sigma Chemical Co. , St. Louis, MO) was added to exponentially growing cultures (1–2 × 106 cells/ml) as described by , except that stocks were stored in aliquots at −80°C, and these were warmed briefly to 50°C immediately before use to ensure complete solubilization.

    Techniques: Mutagenesis, Staining

    Smy1p caps can form in cells lacking microtubules. cdc4 / cdc4 diploid cells (strain 314D5) growing exponentially in YM-P at 24°C were shifted to restrictive temperature (36°C) and incubated for 120 min. By this time, most cells had produced one or two abnormally elongated buds. 15 μg/ml nocodazole ( closed symbols ) or carrier DMSO alone ( open symbols ) was added, and after an additional 45 min of incubation, cells were osmotically shocked (at 0 h) by addition of 0.4 M NaCl (using a 5-M stock). At each time point, samples were processed for indirect immunofluorescence microscopy, and at least 200 cells were scored for the presence of cytoplasmic microtubules ( triangles ) or Smy1p caps ( circles ). As observed previously, nocodazole treatment abolished most cytoplasmic microtubules in

    Journal: The Journal of Cell Biology

    Article Title: Smy1p, a Kinesin-related Protein That Does Not Require Microtubules

    doi:

    Figure Lengend Snippet: Smy1p caps can form in cells lacking microtubules. cdc4 / cdc4 diploid cells (strain 314D5) growing exponentially in YM-P at 24°C were shifted to restrictive temperature (36°C) and incubated for 120 min. By this time, most cells had produced one or two abnormally elongated buds. 15 μg/ml nocodazole ( closed symbols ) or carrier DMSO alone ( open symbols ) was added, and after an additional 45 min of incubation, cells were osmotically shocked (at 0 h) by addition of 0.4 M NaCl (using a 5-M stock). At each time point, samples were processed for indirect immunofluorescence microscopy, and at least 200 cells were scored for the presence of cytoplasmic microtubules ( triangles ) or Smy1p caps ( circles ). As observed previously, nocodazole treatment abolished most cytoplasmic microtubules in

    Article Snippet: Nocodazole Treatment and Immunofluorescence Nocodazole ( Sigma Chemical Co. , St. Louis, MO) was added to exponentially growing cultures (1–2 × 106 cells/ml) as described by , except that stocks were stored in aliquots at −80°C, and these were warmed briefly to 50°C immediately before use to ensure complete solubilization.

    Techniques: Incubation, Produced, Immunofluorescence, Microscopy

    SMY1 -dependent bud growth in the myo2 mutant at restrictive temperature does not require microtubules. ( a ) Nocodazole causes arrest at the large-budded stage of the cell cycle because mitosis but not bud growth is blocked ( Jacobs et al., 1988 ), whereas the myo2 mutation interferes with bud growth giving rise to abnormally large mother cells ( Johnston et al., 1991 ). Counts were made of large-budded ( b ) and unbudded ( c ) cells in cultures of the myo2 mutant (strain SLY334) carrying multicopy SMY1 (YEpSMY1-52; circles ) or control vector (YEp352; triangles ). Cells growing in selective medium at 25°C were shifted to restrictive temperature (31°C) by a fivefold dilution into prewarmed rich medium (YM-P) (refer to Materials and Methods). 5 μg/ml nocodazole ( closed symbols ) or carrier (DMSO) alone ( open symbols ) was added 1 h later (at the time indicated by the arrow), to allow time for the myo2 mutation to be “expressed” and for recovery from the transient effects of the temperature shift ( Lillie and Brown, 1994 ). At each time point, samples were fixed and an aliquot was counted after sonication to disperse clumps of cells. For each sample, at least 200 cells were scored as unbudded, small budded, or large budded (cells whose buds were more than three quarters the size of the mother cell). A second aliquot was processed for microtubule staining. In nocodazole-treated cells, all detectable cytoplasmic microtubules had disappeared by 30 min after nocodazole addition although putative spindle pole bodies persisted in many cells and short spindles persisted in an occasional cell. At 2 1 / 2 h, cytoplasmic microtubules began to reappear in some cells, concomitant with an increase in spindle pole body staining. In mock-treated (DMSO carrier alone) cultures, cytoplasmic microtubules (and spindle pole bodies) were detectable in virtually all cells throughout the experiment, while spindles were present in a fraction of cells.

    Journal: The Journal of Cell Biology

    Article Title: Smy1p, a Kinesin-related Protein That Does Not Require Microtubules

    doi:

    Figure Lengend Snippet: SMY1 -dependent bud growth in the myo2 mutant at restrictive temperature does not require microtubules. ( a ) Nocodazole causes arrest at the large-budded stage of the cell cycle because mitosis but not bud growth is blocked ( Jacobs et al., 1988 ), whereas the myo2 mutation interferes with bud growth giving rise to abnormally large mother cells ( Johnston et al., 1991 ). Counts were made of large-budded ( b ) and unbudded ( c ) cells in cultures of the myo2 mutant (strain SLY334) carrying multicopy SMY1 (YEpSMY1-52; circles ) or control vector (YEp352; triangles ). Cells growing in selective medium at 25°C were shifted to restrictive temperature (31°C) by a fivefold dilution into prewarmed rich medium (YM-P) (refer to Materials and Methods). 5 μg/ml nocodazole ( closed symbols ) or carrier (DMSO) alone ( open symbols ) was added 1 h later (at the time indicated by the arrow), to allow time for the myo2 mutation to be “expressed” and for recovery from the transient effects of the temperature shift ( Lillie and Brown, 1994 ). At each time point, samples were fixed and an aliquot was counted after sonication to disperse clumps of cells. For each sample, at least 200 cells were scored as unbudded, small budded, or large budded (cells whose buds were more than three quarters the size of the mother cell). A second aliquot was processed for microtubule staining. In nocodazole-treated cells, all detectable cytoplasmic microtubules had disappeared by 30 min after nocodazole addition although putative spindle pole bodies persisted in many cells and short spindles persisted in an occasional cell. At 2 1 / 2 h, cytoplasmic microtubules began to reappear in some cells, concomitant with an increase in spindle pole body staining. In mock-treated (DMSO carrier alone) cultures, cytoplasmic microtubules (and spindle pole bodies) were detectable in virtually all cells throughout the experiment, while spindles were present in a fraction of cells.

    Article Snippet: Nocodazole Treatment and Immunofluorescence Nocodazole ( Sigma Chemical Co. , St. Louis, MO) was added to exponentially growing cultures (1–2 × 106 cells/ml) as described by , except that stocks were stored in aliquots at −80°C, and these were warmed briefly to 50°C immediately before use to ensure complete solubilization.

    Techniques: Mutagenesis, Plasmid Preparation, Sonication, Staining

    Smy1p caps are present in cells lacking microtubules. Immunolocalization of microtubules ( a and b ) and Smy1p ( c and d ) in the multibudded cdc4 mutant (strain 314D5) in the absence ( a and c ) or presence ( b and d ) of 15 μg/ml nocodazole. cdc4 / cdc4 diploid cells growing exponentially in YM-P at 24°C were shifted to restrictive temperature (36°C) and incubated for 165 min. By this time, most cells had produced one or two abnormally elongated buds. Nocodazole or carrier DMSO alone was added, and samples were processed for indirect immunofluorescence microscopy at 60, 95, and 120 min. 200 cells/time point were scored for the presence of Smy1p caps, Myo2p caps, and microtubules; results were indistinguishable at these three time points (see text for the ranges). As expected ( Jacobs et al., 1988 ), virtually every bud contained a prominent bundle of microtubules in the absence ( a ) but not the presence ( b ) of nocodazole. The small dots seen in b are caused by residual staining of the spindle pole bodies. A subset of the buds contained Smy1p caps (and Myo2p caps; data not shown) whether ( d ) or not ( c ) nocodazole was present. The arrow in d indicates a (putatively) newly forming bud with a bright Smy1p cap. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Smy1p, a Kinesin-related Protein That Does Not Require Microtubules

    doi:

    Figure Lengend Snippet: Smy1p caps are present in cells lacking microtubules. Immunolocalization of microtubules ( a and b ) and Smy1p ( c and d ) in the multibudded cdc4 mutant (strain 314D5) in the absence ( a and c ) or presence ( b and d ) of 15 μg/ml nocodazole. cdc4 / cdc4 diploid cells growing exponentially in YM-P at 24°C were shifted to restrictive temperature (36°C) and incubated for 165 min. By this time, most cells had produced one or two abnormally elongated buds. Nocodazole or carrier DMSO alone was added, and samples were processed for indirect immunofluorescence microscopy at 60, 95, and 120 min. 200 cells/time point were scored for the presence of Smy1p caps, Myo2p caps, and microtubules; results were indistinguishable at these three time points (see text for the ranges). As expected ( Jacobs et al., 1988 ), virtually every bud contained a prominent bundle of microtubules in the absence ( a ) but not the presence ( b ) of nocodazole. The small dots seen in b are caused by residual staining of the spindle pole bodies. A subset of the buds contained Smy1p caps (and Myo2p caps; data not shown) whether ( d ) or not ( c ) nocodazole was present. The arrow in d indicates a (putatively) newly forming bud with a bright Smy1p cap. Bar, 10 μm.

    Article Snippet: Nocodazole Treatment and Immunofluorescence Nocodazole ( Sigma Chemical Co. , St. Louis, MO) was added to exponentially growing cultures (1–2 × 106 cells/ml) as described by , except that stocks were stored in aliquots at −80°C, and these were warmed briefly to 50°C immediately before use to ensure complete solubilization.

    Techniques: Mutagenesis, Incubation, Produced, Immunofluorescence, Microscopy, Staining

    DY131 delays chromosome segregation in mitosis A. Individual frames representative of prophase, metaphase, and anaphase/telophase from live-cell confocal microscopy of MCF7 cells stably expressing GFP-H2B. Cells were accumulated in G2 by exposure to nocodazole, then released into DY131 or DMSO control. Arrows denote cells of interest. B. Quantitation of time elapsed from chromatin condensation (prophase) to anaphase in MCF7 cells stably expressing GFP-H2B after release from nocodazole block into DY131 or DMSO control. N = 4 – 11 cells, one-way ANOVA with Tukey's post-test.

    Journal: Oncotarget

    Article Title: Antimitotic activity of DY131 and the estrogen-related receptor beta 2 (ERRβ2) splice variant in breast cancer

    doi: 10.18632/oncotarget.9719

    Figure Lengend Snippet: DY131 delays chromosome segregation in mitosis A. Individual frames representative of prophase, metaphase, and anaphase/telophase from live-cell confocal microscopy of MCF7 cells stably expressing GFP-H2B. Cells were accumulated in G2 by exposure to nocodazole, then released into DY131 or DMSO control. Arrows denote cells of interest. B. Quantitation of time elapsed from chromatin condensation (prophase) to anaphase in MCF7 cells stably expressing GFP-H2B after release from nocodazole block into DY131 or DMSO control. N = 4 – 11 cells, one-way ANOVA with Tukey's post-test.

    Article Snippet: The microtubule inhibitor nocodazole (Sigma Aldrich), Smoothened inhibitors vismodegib and cyclopamine (kind gifts from Dr. Insoo Bae), paclitaxel (generously provided by Dr. Robert Clarke), flavopiridol (kind gift from Dr. Christopher Albanese), and the ATM inhibitor KU-55933 (generously provided by Dr. Gil Palchik) were also prepared as concentrated stocks in DMSO, stored at −20°C or 4°C (nocodazole), and used at the indicated concentrations.

    Techniques: Confocal Microscopy, Stable Transfection, Expressing, Quantitation Assay, Blocking Assay

    Actin polymerization signaling pathways are required for TNT formation in RAW/LR5 macrophages. ( a ) Montage of time-lapse live images of GFP-CAAX RAW/LR5 macrophages showing the formation of a TNT-like protrusion (indicated by the yellow arrow) that then connects to an adjacent cell. For visualization of fine less intense structures within the narrow dynamic range available for display, images were local contrast enhanced. Scale bar: 10 µm. ( b ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, actin polymerization inhibitor cytochalasin D (2 μM), or microtubule polymerization inhibitor Nocodazole (2 μM). TNT formation was quantified 4 hours after treatment. The number of TNT connections is represented as in (2d). ( c ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, Arp2/3 inhibitor CK666 (40 μM), Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10 μM). TNT formation was quantified 4 hours after treatment. ( d ) Following quick adherence, bone marrow derived macrophages (BMMs) isolated from 3 independent mice were treated with vehicle control, Arp2/3 inhibitor CK666 (40 μM), or actin polymerization inhibitor cytochalasin D (2 μM). TNT formation was quantified 4 hours after treatment. ( e ) Following quick adherence, BMMs isolated from 3 independent mice were treated with vehicle control, Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10μM). TNT formation was quantified 4 hours after treatment. Data in all graphs is represented as dot plots showing the individual values of the number of TNT connections for each independent experiment in each case. The outlined histograms represent the mean average of at least 3 independent experiments. Error bars +/−SEM with *p

    Journal: Scientific Reports

    Article Title: The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis

    doi: 10.1038/s41598-017-08950-7

    Figure Lengend Snippet: Actin polymerization signaling pathways are required for TNT formation in RAW/LR5 macrophages. ( a ) Montage of time-lapse live images of GFP-CAAX RAW/LR5 macrophages showing the formation of a TNT-like protrusion (indicated by the yellow arrow) that then connects to an adjacent cell. For visualization of fine less intense structures within the narrow dynamic range available for display, images were local contrast enhanced. Scale bar: 10 µm. ( b ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, actin polymerization inhibitor cytochalasin D (2 μM), or microtubule polymerization inhibitor Nocodazole (2 μM). TNT formation was quantified 4 hours after treatment. The number of TNT connections is represented as in (2d). ( c ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, Arp2/3 inhibitor CK666 (40 μM), Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10 μM). TNT formation was quantified 4 hours after treatment. ( d ) Following quick adherence, bone marrow derived macrophages (BMMs) isolated from 3 independent mice were treated with vehicle control, Arp2/3 inhibitor CK666 (40 μM), or actin polymerization inhibitor cytochalasin D (2 μM). TNT formation was quantified 4 hours after treatment. ( e ) Following quick adherence, BMMs isolated from 3 independent mice were treated with vehicle control, Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10μM). TNT formation was quantified 4 hours after treatment. Data in all graphs is represented as dot plots showing the individual values of the number of TNT connections for each independent experiment in each case. The outlined histograms represent the mean average of at least 3 independent experiments. Error bars +/−SEM with *p

    Article Snippet: Microtubule inhibitors Nocodazole (Sigma) was used at 2 µM.

    Techniques: Incubation, Derivative Assay, Isolation, Mouse Assay

    Chromosome length as a parameter for metaphase spread quality for karyotype analysis. The average length of chromosome 1 (here with the centromeric region marked in red) was measured using the image analysis package MetaMorph v7.6 (example above), and compared between the nocodazole 16 h/buffered hypotonic harvest and the demecolcine 16 h/buffered hypotonic harvest

    Journal: Stem Cell Reviews

    Article Title: An Improved Technique for Chromosomal Analysis of Human ES and iPS Cells

    doi: 10.1007/s12015-010-9224-4

    Figure Lengend Snippet: Chromosome length as a parameter for metaphase spread quality for karyotype analysis. The average length of chromosome 1 (here with the centromeric region marked in red) was measured using the image analysis package MetaMorph v7.6 (example above), and compared between the nocodazole 16 h/buffered hypotonic harvest and the demecolcine 16 h/buffered hypotonic harvest

    Article Snippet: Chromosome Harvest Nocodazole (Sigma Aldrich, U.K., http://www.sigmaaldrich.com ), 2.5 mg/ml stock solution in dimethyl sulfoxide, (DMSO) (Sigma Aldrich, U.K) (Note 1).

    Techniques:

    Efficiency of mitotic arrest following treatment with either demecolcine or nocodazole. Different concentrations and incubation times were compared. After fixation the cells were stained with DAPI, and analyzed at the microscope. Ten random fields from each of the slides prepared under different conditions were captured with Genus on the CytoVision system. The yellow arrows identify metaphasic cells

    Journal: Stem Cell Reviews

    Article Title: An Improved Technique for Chromosomal Analysis of Human ES and iPS Cells

    doi: 10.1007/s12015-010-9224-4

    Figure Lengend Snippet: Efficiency of mitotic arrest following treatment with either demecolcine or nocodazole. Different concentrations and incubation times were compared. After fixation the cells were stained with DAPI, and analyzed at the microscope. Ten random fields from each of the slides prepared under different conditions were captured with Genus on the CytoVision system. The yellow arrows identify metaphasic cells

    Article Snippet: Chromosome Harvest Nocodazole (Sigma Aldrich, U.K., http://www.sigmaaldrich.com ), 2.5 mg/ml stock solution in dimethyl sulfoxide, (DMSO) (Sigma Aldrich, U.K) (Note 1).

    Techniques: Incubation, Staining, Microscopy

    Stabilization of microtubules at focal adhesions. (A–D ) Figure shows a 3T3 fibroblast that was fixed and triple labeled for actin ( D ), paxillin ( B ), and tubulin ( A and C ) after treatment with 1.5 μg/ml nocodazole for 10 min. All peripheral microtubules disassembled, except those whose ends targeted focal adhesions ( arrowheads ). ( E ) Video sequence showing the stabilization of a shrinking microtubule at a focal adhesion. Goldfish fibroblast co- injected with vinculin and tubulin. Frames are taken from a video sequence for which nocodazole (1.5 μg/ ml) was added at time 0. One of a pair of microtubules that extended to the periphery at the beginning of the sequence ( white arrowhead ) was prevented from shrinking beyond an adhesion site over which it passed ( arrow ). Eventually, it shrank into this adhesion site via depolymerization at its minus end ( black arrowhead ). Bars, 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: Targeting, Capture, and Stabilization of Microtubules at Early Focal Adhesions

    doi:

    Figure Lengend Snippet: Stabilization of microtubules at focal adhesions. (A–D ) Figure shows a 3T3 fibroblast that was fixed and triple labeled for actin ( D ), paxillin ( B ), and tubulin ( A and C ) after treatment with 1.5 μg/ml nocodazole for 10 min. All peripheral microtubules disassembled, except those whose ends targeted focal adhesions ( arrowheads ). ( E ) Video sequence showing the stabilization of a shrinking microtubule at a focal adhesion. Goldfish fibroblast co- injected with vinculin and tubulin. Frames are taken from a video sequence for which nocodazole (1.5 μg/ ml) was added at time 0. One of a pair of microtubules that extended to the periphery at the beginning of the sequence ( white arrowhead ) was prevented from shrinking beyond an adhesion site over which it passed ( arrow ). Eventually, it shrank into this adhesion site via depolymerization at its minus end ( black arrowhead ). Bars, 5 μm.

    Article Snippet: Microtubule Antagonists Nocodazole ( Sigma Chemical Co. ) was added to culture medium from a 5 mg/ml stock solution in DMSO.

    Techniques: Labeling, Sequencing, Injection

    ( A and B ) Targeting, but not stabilization at focal complexes. Images of porcine testicular cells labeled for tubulin and vinculin: A , control cell; B , cell treated with 1.5 μg/ml nocodazole for 20 min. The focal complexes characteristically found on the edges of these cells are targeted by microtubules ( A ), but they do not stabilize microtubules against depolymerization by nocodazole ( B ). ( C and D ) Targeting and stabilization occurs in the absence of intermediate filaments. Fibroblasts of a mouse vimentin knockout cell line labeled for tubulin and vinculin. C , control cell showing targeting of microtubules to contact sites; D , cell treated with 2.5 μg/ml nocodazole for 10 min showing stabilization of microtubules at focal contacts. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Targeting, Capture, and Stabilization of Microtubules at Early Focal Adhesions

    doi:

    Figure Lengend Snippet: ( A and B ) Targeting, but not stabilization at focal complexes. Images of porcine testicular cells labeled for tubulin and vinculin: A , control cell; B , cell treated with 1.5 μg/ml nocodazole for 20 min. The focal complexes characteristically found on the edges of these cells are targeted by microtubules ( A ), but they do not stabilize microtubules against depolymerization by nocodazole ( B ). ( C and D ) Targeting and stabilization occurs in the absence of intermediate filaments. Fibroblasts of a mouse vimentin knockout cell line labeled for tubulin and vinculin. C , control cell showing targeting of microtubules to contact sites; D , cell treated with 2.5 μg/ml nocodazole for 10 min showing stabilization of microtubules at focal contacts. Bar, 10 μm.

    Article Snippet: Microtubule Antagonists Nocodazole ( Sigma Chemical Co. ) was added to culture medium from a 5 mg/ml stock solution in DMSO.

    Techniques: Labeling, Knock-Out

    General stabilization of microtubules by focal adhesions in REF-52 fibroblasts. Cells spreading on fibronectin show numerous focal adhesions ( A ) as compared with a finely punctate vinculin label on polylysine ( E ). The corresponding microtubule distributions are shown in B and F . After brief nocodazole treatment (1.5 μg/ ml, 10 min) peripheral microtubules in cells plated on fibronectin remain essentially unaffected ( D ) whereas those in cells spread on polylysine shrink rapidly into the cell body ( H ). Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Targeting, Capture, and Stabilization of Microtubules at Early Focal Adhesions

    doi:

    Figure Lengend Snippet: General stabilization of microtubules by focal adhesions in REF-52 fibroblasts. Cells spreading on fibronectin show numerous focal adhesions ( A ) as compared with a finely punctate vinculin label on polylysine ( E ). The corresponding microtubule distributions are shown in B and F . After brief nocodazole treatment (1.5 μg/ ml, 10 min) peripheral microtubules in cells plated on fibronectin remain essentially unaffected ( D ) whereas those in cells spread on polylysine shrink rapidly into the cell body ( H ). Bar, 10 μm.

    Article Snippet: Microtubule Antagonists Nocodazole ( Sigma Chemical Co. ) was added to culture medium from a 5 mg/ml stock solution in DMSO.

    Techniques:

    Capture and stabilization of a microtubule at a focal adhesion that was remote from the contact site at the time of addition of nocodazole. Conditions as for Fig. 4 , except that negative and positive times signify before and after nocodazole addition, respectively. Before nocodazole treatment, the microtubule marked with an arrowhead grew and moved laterally and became positioned over a focal adhesion ( arrow , 0′44′′ ). Nocodazole caused rapid shrinkage down to the contact (+ 1′42′′ –2′16′′ ), where the end then remained stable for a further 3 min ( 2′16′′ –5′06′′ ) before finally shrinking into the cell body ( 5′23′′– 6′31′′ ). Bar, 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: Targeting, Capture, and Stabilization of Microtubules at Early Focal Adhesions

    doi:

    Figure Lengend Snippet: Capture and stabilization of a microtubule at a focal adhesion that was remote from the contact site at the time of addition of nocodazole. Conditions as for Fig. 4 , except that negative and positive times signify before and after nocodazole addition, respectively. Before nocodazole treatment, the microtubule marked with an arrowhead grew and moved laterally and became positioned over a focal adhesion ( arrow , 0′44′′ ). Nocodazole caused rapid shrinkage down to the contact (+ 1′42′′ –2′16′′ ), where the end then remained stable for a further 3 min ( 2′16′′ –5′06′′ ) before finally shrinking into the cell body ( 5′23′′– 6′31′′ ). Bar, 5 μm.

    Article Snippet: Microtubule Antagonists Nocodazole ( Sigma Chemical Co. ) was added to culture medium from a 5 mg/ml stock solution in DMSO.

    Techniques:

    Nucleation of microtubule growth at focal adhesions, in 3T3 fibroblasts. A and B show a 3T3 fibroblast labeled for actin and tubulin that had been exposed to 0.05 μm taxol for 1 h before fixation. Arrowheads indicate the ends of some of the microtubules that had been nucleated at the stress fiber terminus ( arrow ). ( C–E ) Part of a REF-52 fibroblast after short term (4 min) recovery from complete disassembly of microtubules by nocodazole. Non-centrosomal microtubule segments ( D ) are specifically associated with peripheral focal adhesions ( C and E ), marked with arrows. In E , the microtubule segments ( D ) have been graphically superimposed on the vinculin image to show the correspondence between the two patterns. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Targeting, Capture, and Stabilization of Microtubules at Early Focal Adhesions

    doi:

    Figure Lengend Snippet: Nucleation of microtubule growth at focal adhesions, in 3T3 fibroblasts. A and B show a 3T3 fibroblast labeled for actin and tubulin that had been exposed to 0.05 μm taxol for 1 h before fixation. Arrowheads indicate the ends of some of the microtubules that had been nucleated at the stress fiber terminus ( arrow ). ( C–E ) Part of a REF-52 fibroblast after short term (4 min) recovery from complete disassembly of microtubules by nocodazole. Non-centrosomal microtubule segments ( D ) are specifically associated with peripheral focal adhesions ( C and E ), marked with arrows. In E , the microtubule segments ( D ) have been graphically superimposed on the vinculin image to show the correspondence between the two patterns. Bar, 10 μm.

    Article Snippet: Microtubule Antagonists Nocodazole ( Sigma Chemical Co. ) was added to culture medium from a 5 mg/ml stock solution in DMSO.

    Techniques: Labeling

    Effect of BFA and nocodazole treatment on TGEV assembly. (A) Large virions (arrowheads) and small dense viral particles (arrows) coexist within the Golgi complex (G) of normal infected cells. (B) BFA causes the disappearance of the Golgi complex as a distinguishable structure, together with the formation of large cisternae (asterisks), some of them apparently derived from the rough endoplasmic reticulum (RER), since they have ribosomes (r) attached. TGEV virions assemble in association with these cisternae (arrowheads point to budding profiles). Large viral particles with an electron-dense internal periphery and clear center (arrows) accumulate in these conditions. Some ERGIC-like tubular membranes are visible in these cells (pairs of arrows). (C) Abnormal Golgi stack (G) from a nocodazole-treated infected ST cell. Both budding profiles (arrowheads) and small dense virions (arrows) are seen within the altered membranes of the stack. mi, mitochondria; pm, plasma membrane. Bar, 200 nm.

    Journal: Journal of Virology

    Article Title: Two Types of Virus-Related Particles Are Found during Transmissible Gastroenteritis Virus Morphogenesis

    doi:

    Figure Lengend Snippet: Effect of BFA and nocodazole treatment on TGEV assembly. (A) Large virions (arrowheads) and small dense viral particles (arrows) coexist within the Golgi complex (G) of normal infected cells. (B) BFA causes the disappearance of the Golgi complex as a distinguishable structure, together with the formation of large cisternae (asterisks), some of them apparently derived from the rough endoplasmic reticulum (RER), since they have ribosomes (r) attached. TGEV virions assemble in association with these cisternae (arrowheads point to budding profiles). Large viral particles with an electron-dense internal periphery and clear center (arrows) accumulate in these conditions. Some ERGIC-like tubular membranes are visible in these cells (pairs of arrows). (C) Abnormal Golgi stack (G) from a nocodazole-treated infected ST cell. Both budding profiles (arrowheads) and small dense virions (arrows) are seen within the altered membranes of the stack. mi, mitochondria; pm, plasma membrane. Bar, 200 nm.

    Article Snippet: Confluent monolayers of ST cells were infected with TGEV at a multiplicity of infection of 10 PFU/cell, and at 4 h p.i., cultures were treated with the microtubular disrupting agent nocodazole (Sigma) at different concentrations (1, 3, 10, and 20 μM) and incubated for 4 h at 37°C.

    Techniques: Infection, Derivative Assay

    CENP-I increases the half-life of Mad1 at unattached kinetochores. (A) Images of Mad1-GFP FRAP in control, CENP-I–depleted, and ZM-treated cells arrested in nocodazole. (B) Recovery dynamics of Mad1-GFP after photobleaching demonstrating that CENP-I–depleted cells have a larger initial recovery of Mad1 and a faster turnover of stable Mad1. (C) Total recovery of Mad1-GFP at 120 s after photobleaching. (D) Scatter plot displaying the natural log of the normalized unrecovered fluorescence over time. The biphasic nature of Mad1 recovery is illustrated by overlaid lines. CENP-I–depleted cells have a fast phase of initial Mad1 recovery similar to controls but the pool of stable Mad1 in CENP-I–depleted cells has a greatly decreased half-life relative to control. Red arrows in A indicate FRAP targets. FRAP data are from n = 30 experiments. Error bars indicate standard deviation. *, P

    Journal: The Journal of Cell Biology

    Article Title: CENP-I and Aurora B act as a molecular switch that ties RZZ/Mad1 recruitment to kinetochore attachment status

    doi: 10.1083/jcb.201307137

    Figure Lengend Snippet: CENP-I increases the half-life of Mad1 at unattached kinetochores. (A) Images of Mad1-GFP FRAP in control, CENP-I–depleted, and ZM-treated cells arrested in nocodazole. (B) Recovery dynamics of Mad1-GFP after photobleaching demonstrating that CENP-I–depleted cells have a larger initial recovery of Mad1 and a faster turnover of stable Mad1. (C) Total recovery of Mad1-GFP at 120 s after photobleaching. (D) Scatter plot displaying the natural log of the normalized unrecovered fluorescence over time. The biphasic nature of Mad1 recovery is illustrated by overlaid lines. CENP-I–depleted cells have a fast phase of initial Mad1 recovery similar to controls but the pool of stable Mad1 in CENP-I–depleted cells has a greatly decreased half-life relative to control. Red arrows in A indicate FRAP targets. FRAP data are from n = 30 experiments. Error bars indicate standard deviation. *, P

    Article Snippet: Nocodazole treatments and nocodazole washout assays Nocodazole (Sigma-Aldrich) was used at 3.3 µM throughout the study, a concentration sufficient to depolymerize all microtubules, and cells were treated for 2 h unless otherwise indicated.

    Techniques: Fluorescence, Standard Deviation

    Centromere Aurora B localization and activity is enhanced by microtubules at kinetochores. (A) Aurora B localizes to all centromeres during prometaphase and in nocodazole. After nocodazole washout, Aurora B specifically localizes to centromeres where kinetochores overlap with microtubules and is reduced or lost at kinetochores without microtubules. (B) After nocodazole washout Aurora B is specifically enhanced at kinetochores with microtubules and is reduced at kinetochores without microtubules. (C) Quantification of Aurora B centromere intensities in nocodazole and after nocodazole washout demonstrating an increase in overall Aurora B staining across all centromeres after nocodazole washout. (D) Aurora B activity as visualized by p(S7)CENP-A phosphorylation in prometaphase, in nocodazole, and after nocodazole washout. Aurora B activity correlates with the presence of microtubules at kinetochores. (E) p(S7)CENP-A phosphorylation levels are high at kinetochores with microtubules and low at kinetochores without microtubules after nocodazole washout. Yellow arrows indicate select examples of kinetochores without detectable microtubules. Blue arrows indicate select examples of kinetochores with associated microtubules. Each image represents multiple Z-slices. Error bars indicate standard deviation. *, P

    Journal: The Journal of Cell Biology

    Article Title: CENP-I and Aurora B act as a molecular switch that ties RZZ/Mad1 recruitment to kinetochore attachment status

    doi: 10.1083/jcb.201307137

    Figure Lengend Snippet: Centromere Aurora B localization and activity is enhanced by microtubules at kinetochores. (A) Aurora B localizes to all centromeres during prometaphase and in nocodazole. After nocodazole washout, Aurora B specifically localizes to centromeres where kinetochores overlap with microtubules and is reduced or lost at kinetochores without microtubules. (B) After nocodazole washout Aurora B is specifically enhanced at kinetochores with microtubules and is reduced at kinetochores without microtubules. (C) Quantification of Aurora B centromere intensities in nocodazole and after nocodazole washout demonstrating an increase in overall Aurora B staining across all centromeres after nocodazole washout. (D) Aurora B activity as visualized by p(S7)CENP-A phosphorylation in prometaphase, in nocodazole, and after nocodazole washout. Aurora B activity correlates with the presence of microtubules at kinetochores. (E) p(S7)CENP-A phosphorylation levels are high at kinetochores with microtubules and low at kinetochores without microtubules after nocodazole washout. Yellow arrows indicate select examples of kinetochores without detectable microtubules. Blue arrows indicate select examples of kinetochores with associated microtubules. Each image represents multiple Z-slices. Error bars indicate standard deviation. *, P

    Article Snippet: Nocodazole treatments and nocodazole washout assays Nocodazole (Sigma-Aldrich) was used at 3.3 µM throughout the study, a concentration sufficient to depolymerize all microtubules, and cells were treated for 2 h unless otherwise indicated.

    Techniques: Activity Assay, Staining, Standard Deviation

    CENP-I–depleted kinetochores lose Mad1 from kinetochores faster than control kinetochores in the presence of microtubules. (A) Control cells retain Mad1 at kinetochores up to 20 min after nocodazole washout. (B) CENP-I–depleted cells lose all Mad1 from kinetochores between 8–12 min after nocodazole washout, even before a bipolar spindle can form. At 16 min, Mad1 can inbriefly be seen at the vertices of microtubule bundles. (C) Quantification of mean Mad1 kinetochore levels across all kinetochores from A and B. Error bars indicate standard deviation. *, P

    Journal: The Journal of Cell Biology

    Article Title: CENP-I and Aurora B act as a molecular switch that ties RZZ/Mad1 recruitment to kinetochore attachment status

    doi: 10.1083/jcb.201307137

    Figure Lengend Snippet: CENP-I–depleted kinetochores lose Mad1 from kinetochores faster than control kinetochores in the presence of microtubules. (A) Control cells retain Mad1 at kinetochores up to 20 min after nocodazole washout. (B) CENP-I–depleted cells lose all Mad1 from kinetochores between 8–12 min after nocodazole washout, even before a bipolar spindle can form. At 16 min, Mad1 can inbriefly be seen at the vertices of microtubule bundles. (C) Quantification of mean Mad1 kinetochore levels across all kinetochores from A and B. Error bars indicate standard deviation. *, P

    Article Snippet: Nocodazole treatments and nocodazole washout assays Nocodazole (Sigma-Aldrich) was used at 3.3 µM throughout the study, a concentration sufficient to depolymerize all microtubules, and cells were treated for 2 h unless otherwise indicated.

    Techniques: Standard Deviation

    CENP-I–depleted kinetochores fail to inhibit dynein-mediated stripping of Mad1. (A) Immunofluorescence images of Mad1 in control and CENP-I–depleted cells 10 min after nocodazole washout, with or without expression of the dynein inhibitor CC1. Control cells retain Mad1 at kinetochores after nocodazole washout, but CENP-I–depleted cells rapidly lose Mad1 from kinetochores and accumulate it at spindle poles in a dynein-dependent manner. (B) Quantification of the total number of Mad1-positive kinetochores in cells from conditions depicted in A. (C) Immunofluorescence images of CENP-I–depleted cells demonstrating that inhibition of dynein does not prevent recruitment of Mad1 to unattached kinetochores, but does prevent loss of Mad1 from kinetochores after nocodazole washout. Centromeres are labeled to demonstrate that Mad1 is at kinetochores. Blue arrows indicate position of spindle poles. Cy5-labeled anti-Mad1 antibody is displayed here in green for ease of viewing. Error bars indicate standard deviation. *, P

    Journal: The Journal of Cell Biology

    Article Title: CENP-I and Aurora B act as a molecular switch that ties RZZ/Mad1 recruitment to kinetochore attachment status

    doi: 10.1083/jcb.201307137

    Figure Lengend Snippet: CENP-I–depleted kinetochores fail to inhibit dynein-mediated stripping of Mad1. (A) Immunofluorescence images of Mad1 in control and CENP-I–depleted cells 10 min after nocodazole washout, with or without expression of the dynein inhibitor CC1. Control cells retain Mad1 at kinetochores after nocodazole washout, but CENP-I–depleted cells rapidly lose Mad1 from kinetochores and accumulate it at spindle poles in a dynein-dependent manner. (B) Quantification of the total number of Mad1-positive kinetochores in cells from conditions depicted in A. (C) Immunofluorescence images of CENP-I–depleted cells demonstrating that inhibition of dynein does not prevent recruitment of Mad1 to unattached kinetochores, but does prevent loss of Mad1 from kinetochores after nocodazole washout. Centromeres are labeled to demonstrate that Mad1 is at kinetochores. Blue arrows indicate position of spindle poles. Cy5-labeled anti-Mad1 antibody is displayed here in green for ease of viewing. Error bars indicate standard deviation. *, P

    Article Snippet: Nocodazole treatments and nocodazole washout assays Nocodazole (Sigma-Aldrich) was used at 3.3 µM throughout the study, a concentration sufficient to depolymerize all microtubules, and cells were treated for 2 h unless otherwise indicated.

    Techniques: Stripping Membranes, Immunofluorescence, Expressing, Inhibition, Labeling, Standard Deviation

    Aurora B activity or CENP-I are required to localize Mad1 and ZW10 to unattached kinetochores. (A) Simplified model depicting how Aurora B and CENP-H/I/K function to localize Mad1 at kinetochores. (B and C) Thymidine release assays demonstrating that either CENP-I or Aurora B activity are able to localize Mad1 and ZW10 to unattached kinetochores at the onset of mitosis. After Aurora B inhibition and CENP-I depletion both Mad1 and ZW10 are not at kinetochores. (D) Quantification of Mad1 and ZW10 kinetochore localization from B and C. All cells were treated with 3.3 µM nocodazole. Selected examples of kinetochores without Mad1 or ZW10 are indicated by yellow arrows. Error bars indicate standard deviation. *, P

    Journal: The Journal of Cell Biology

    Article Title: CENP-I and Aurora B act as a molecular switch that ties RZZ/Mad1 recruitment to kinetochore attachment status

    doi: 10.1083/jcb.201307137

    Figure Lengend Snippet: Aurora B activity or CENP-I are required to localize Mad1 and ZW10 to unattached kinetochores. (A) Simplified model depicting how Aurora B and CENP-H/I/K function to localize Mad1 at kinetochores. (B and C) Thymidine release assays demonstrating that either CENP-I or Aurora B activity are able to localize Mad1 and ZW10 to unattached kinetochores at the onset of mitosis. After Aurora B inhibition and CENP-I depletion both Mad1 and ZW10 are not at kinetochores. (D) Quantification of Mad1 and ZW10 kinetochore localization from B and C. All cells were treated with 3.3 µM nocodazole. Selected examples of kinetochores without Mad1 or ZW10 are indicated by yellow arrows. Error bars indicate standard deviation. *, P

    Article Snippet: Nocodazole treatments and nocodazole washout assays Nocodazole (Sigma-Aldrich) was used at 3.3 µM throughout the study, a concentration sufficient to depolymerize all microtubules, and cells were treated for 2 h unless otherwise indicated.

    Techniques: Activity Assay, Inhibition, Standard Deviation

    Microtubules trigger Mad1 recruitment to individual kinetochores in CENP-I–depleted cells. (A) Control-depleted cells localize Mad1 to kinetochores in prometaphase, during nocodazole treatment, and 10 min after nocodazole washout. CENP-I–depleted cells have no Mad1 at kinetochores in prometaphase but can fully recruit Mad1 to kinetochores in nocodazole. After nocodazole washout, Mad1 is specifically recruited to kinetochores that overlap with microtubules and is absent from kinetochores without microtubules. (B) Quantification of Mad1 kinetochore intensities from A showing that CENP-I–depleted cells fully recruit Mad1 to unattached kinetochores in nocodazole but lose most Mad1 from kinetochores after nocodazole washout. (C) Quantification of Mad1 intensities at kinetochores with or without microtubules after nocodazole washout. Control cells have equal amounts of Mad1 at kinetochores with or without microtubules, whereas CENP-I–depleted cells have fivefold more Mad1 at kinetochores with microtubules. Yellow arrows indicate select examples of kinetochores without microtubules. Blue arrows indicate select examples of kinetochores with associated microtubules. Insets contain multiple Z-sections for clarity. Error bars indicate standard deviation. *, P

    Journal: The Journal of Cell Biology

    Article Title: CENP-I and Aurora B act as a molecular switch that ties RZZ/Mad1 recruitment to kinetochore attachment status

    doi: 10.1083/jcb.201307137

    Figure Lengend Snippet: Microtubules trigger Mad1 recruitment to individual kinetochores in CENP-I–depleted cells. (A) Control-depleted cells localize Mad1 to kinetochores in prometaphase, during nocodazole treatment, and 10 min after nocodazole washout. CENP-I–depleted cells have no Mad1 at kinetochores in prometaphase but can fully recruit Mad1 to kinetochores in nocodazole. After nocodazole washout, Mad1 is specifically recruited to kinetochores that overlap with microtubules and is absent from kinetochores without microtubules. (B) Quantification of Mad1 kinetochore intensities from A showing that CENP-I–depleted cells fully recruit Mad1 to unattached kinetochores in nocodazole but lose most Mad1 from kinetochores after nocodazole washout. (C) Quantification of Mad1 intensities at kinetochores with or without microtubules after nocodazole washout. Control cells have equal amounts of Mad1 at kinetochores with or without microtubules, whereas CENP-I–depleted cells have fivefold more Mad1 at kinetochores with microtubules. Yellow arrows indicate select examples of kinetochores without microtubules. Blue arrows indicate select examples of kinetochores with associated microtubules. Insets contain multiple Z-sections for clarity. Error bars indicate standard deviation. *, P

    Article Snippet: Nocodazole treatments and nocodazole washout assays Nocodazole (Sigma-Aldrich) was used at 3.3 µM throughout the study, a concentration sufficient to depolymerize all microtubules, and cells were treated for 2 h unless otherwise indicated.

    Techniques: Standard Deviation

    Cell and nuclear morphology under DMSO and nocodazole treatment. (A) Immunofluorescence images of untreated (this is same example as shown in A), DMSO control and nocodazole-treated MEF cells. Solid and open arrowheads indicate organized and disrupted

    Journal: Journal of Cell Science

    Article Title: Volume regulation and shape bifurcation in the cell nucleus

    doi: 10.1242/jcs.166330

    Figure Lengend Snippet: Cell and nuclear morphology under DMSO and nocodazole treatment. (A) Immunofluorescence images of untreated (this is same example as shown in A), DMSO control and nocodazole-treated MEF cells. Solid and open arrowheads indicate organized and disrupted

    Article Snippet: The microtubule-depolymerizing drug nocodazole (Sigma) was diluted to a final concentration of 1 μM by using the stock solution.

    Techniques: Immunofluorescence

    Formation of v-ErbA foci is microtubule-and dynein-dependent. (A) Treatment with nocodozole disrupts microtubules. HeLa cells were either left untreated, treated with DMSO (vehicle control), or treated with nocodozole (+NOC) for 20 h. Microtubules were visualized by immunostaining with Cy3-tagged anti-β-tubulin (red). Nuclei were stained for DNA with DAPI (blue) (B) Microtubule disruption prevents the formation of coalesced v-ErbA foci. HeLa cells were transfected with an expression vector for GFP-v-ErbA, and 16 h post-transfection were treated with nocodazole for 20 h. +NOC (diffuse), nocodazole-treated cell forming diffuse aggregates; +NOC (small aggregates), nocodazole-treated cell forming small aggregates, uniform in size; −NOC, untreated cell forming large juxtanuclear foci. (C) Bar graph summarizing the effect of nocodozole on v-ErbA foci size. Upon nocodazole treatment, there was a significant shift (p

    Journal: Molecular and cellular endocrinology

    Article Title: Recruitment of the Oncoprotein v-ErbA to Aggresomes

    doi: 10.1016/j.mce.2010.10.012

    Figure Lengend Snippet: Formation of v-ErbA foci is microtubule-and dynein-dependent. (A) Treatment with nocodozole disrupts microtubules. HeLa cells were either left untreated, treated with DMSO (vehicle control), or treated with nocodozole (+NOC) for 20 h. Microtubules were visualized by immunostaining with Cy3-tagged anti-β-tubulin (red). Nuclei were stained for DNA with DAPI (blue) (B) Microtubule disruption prevents the formation of coalesced v-ErbA foci. HeLa cells were transfected with an expression vector for GFP-v-ErbA, and 16 h post-transfection were treated with nocodazole for 20 h. +NOC (diffuse), nocodazole-treated cell forming diffuse aggregates; +NOC (small aggregates), nocodazole-treated cell forming small aggregates, uniform in size; −NOC, untreated cell forming large juxtanuclear foci. (C) Bar graph summarizing the effect of nocodozole on v-ErbA foci size. Upon nocodazole treatment, there was a significant shift (p

    Article Snippet: In some experiments, cells were treated with 2–4 nM leptomycin B (Sigma) or vehicle (methanol) for 1–5 h, 100 nM 3,3′,5-triiodo-L-thyronine (T3 ) (Sigma), the proteasome-inhibiting drug MG132 (10 μg/ml, Sigma; or 10 μM, Calbiochem), the microtubule-disrupting drug nocodazole (10 μg/ml, Sigma), or an equivalent volume of vehicle (DMSO) for 12–20 hr post-transfection.

    Techniques: Immunostaining, Staining, Transfection, Expressing, Plasmid Preparation

    Membrane nanotubes of ARPE -19 cells contained F-actin but not microtubules. Fluorescence image of ARPE-19 cells stained with phalloidin-TRITC for actin (A, B) anti-ß-tubulin (C, D) and nucleus (DAPI). Images represent the cytoskeleton in control (no treatment A, C, E) and after treatment with 15 µM nocodazole for 24 h (B, D, F). (A, B) Fluorescence image of F-actin. Actin fibres were visible in the cells and clearly seen is a straight connection, representing the TNT structure, between the cells (arrows). (C, D) Fluorescence image of ARPE-19 cells stained with mAb against β-tubulin revealed the lack of β-tubulin in the TNTs (arrows). (E, F) Merged pictures with actin (red), ß-tubulin (green) and nucleus (blue). Arrows mark TNTs. Scale bar, 20 µm.

    Journal: PLoS ONE

    Article Title: Multi-Level Communication of Human Retinal Pigment Epithelial Cells via Tunneling Nanotubes

    doi: 10.1371/journal.pone.0033195

    Figure Lengend Snippet: Membrane nanotubes of ARPE -19 cells contained F-actin but not microtubules. Fluorescence image of ARPE-19 cells stained with phalloidin-TRITC for actin (A, B) anti-ß-tubulin (C, D) and nucleus (DAPI). Images represent the cytoskeleton in control (no treatment A, C, E) and after treatment with 15 µM nocodazole for 24 h (B, D, F). (A, B) Fluorescence image of F-actin. Actin fibres were visible in the cells and clearly seen is a straight connection, representing the TNT structure, between the cells (arrows). (C, D) Fluorescence image of ARPE-19 cells stained with mAb against β-tubulin revealed the lack of β-tubulin in the TNTs (arrows). (E, F) Merged pictures with actin (red), ß-tubulin (green) and nucleus (blue). Arrows mark TNTs. Scale bar, 20 µm.

    Article Snippet: To investigate microtubules in TNTs, the microtubule-destabilizing drug nocodazole (Sigma-Aldrich) was added to the medium at a final concentration of 15 µM.

    Techniques: Fluorescence, Staining

    Colocalization of Tctex-1 and Rab3D with microtubules in osteoclasts and effect of nocodazole on Tctex-1–Rab3D interaction in vivo . Osteoclasts were briefly pre-extracted with microtubule stabilizing buffer, fixed in methanol, and then immunostained with the indicated antibodies. (A) A projected XY image stack of an osteoclast stained with anti-Tctex-1 and α-tubulin. (B) A single optical section of an osteoclast stained for anti-Rab3D and α-tubulin. Arrows depict Rab3D-bearing vesicles lying along individual microtubule filaments in magnified insets. (C) Treatment with the microtubule depolymerizing agent nocodazole (2 h) disrupts Rab3D–Tctex-1 distribution in osteoclasts. (D) Effect of brefeldin A, cytochalasin D (cyto D), and nocodazole on Tctex-1–Rab3D interaction as monitored by BRET (***, P

    Journal: Molecular and Cellular Biology

    Article Title: Tctex-1, a Novel Interaction Partner of Rab3D, Is Required for Osteoclastic Bone Resorption ▿

    doi: 10.1128/MCB.00834-10

    Figure Lengend Snippet: Colocalization of Tctex-1 and Rab3D with microtubules in osteoclasts and effect of nocodazole on Tctex-1–Rab3D interaction in vivo . Osteoclasts were briefly pre-extracted with microtubule stabilizing buffer, fixed in methanol, and then immunostained with the indicated antibodies. (A) A projected XY image stack of an osteoclast stained with anti-Tctex-1 and α-tubulin. (B) A single optical section of an osteoclast stained for anti-Rab3D and α-tubulin. Arrows depict Rab3D-bearing vesicles lying along individual microtubule filaments in magnified insets. (C) Treatment with the microtubule depolymerizing agent nocodazole (2 h) disrupts Rab3D–Tctex-1 distribution in osteoclasts. (D) Effect of brefeldin A, cytochalasin D (cyto D), and nocodazole on Tctex-1–Rab3D interaction as monitored by BRET (***, P

    Article Snippet: Where indicated, osteoclasts were incubated with media containing either vehicle (DMSO) or the microtubule depolymerizing agent nocodazole (6 μg/ml; Sigma) for 2 h. Hoechst 3342 dye (Invitrogen) was used to visualize cell nuclei.

    Techniques: In Vivo, Staining, Bioluminescence Resonance Energy Transfer

    Tri- and tetra-radial chromosomes and telomere fusion in U698 and JVM-2 cells. Cells were grown in the absence, or presence of 10 µM KU-55933 and 3 µM Olaparib for 48 h. Nocodazole was added for the last 6 h of incubation to increase the yield of metaphases. Metaphase spreads were prepared by standard cytogenetic methods. Tri- and tetra-radial chromosomes are indicated by filled arrows, telomere fusion is indicated by an asterix.

    Journal: Cell Cycle

    Article Title: Replication-induced DNA damage after PARP inhibition causes G2 delay, and cell line-dependent apoptosis, necrosis and multinucleation

    doi: 10.1080/15384101.2015.1085137

    Figure Lengend Snippet: Tri- and tetra-radial chromosomes and telomere fusion in U698 and JVM-2 cells. Cells were grown in the absence, or presence of 10 µM KU-55933 and 3 µM Olaparib for 48 h. Nocodazole was added for the last 6 h of incubation to increase the yield of metaphases. Metaphase spreads were prepared by standard cytogenetic methods. Tri- and tetra-radial chromosomes are indicated by filled arrows, telomere fusion is indicated by an asterix.

    Article Snippet: To assess the flux of cells into mitosis we employed the microtubule polymerization-inhibitor nocodazole (1ug/ml; Sigma-Aldrich) for 6 h before harvest.

    Techniques: Incubation

    Bub3 depletion abrogates the spindle assembly checkpoint and causes chromosome misalignment. (A) Mitotic index of cells transfected with mock, Bub3, Bub1, or BubR1 siRNAs, before or after treatment with nocodazole. (B) Bub3-depleted cells exhibit abnormal

    Journal: Molecular Biology of the Cell

    Article Title: The Human Spindle Assembly Checkpoint Protein Bub3 Is Required for the Establishment of Efficient Kinetochore-Microtubule Attachments

    doi: 10.1091/mbc.E07-07-0633

    Figure Lengend Snippet: Bub3 depletion abrogates the spindle assembly checkpoint and causes chromosome misalignment. (A) Mitotic index of cells transfected with mock, Bub3, Bub1, or BubR1 siRNAs, before or after treatment with nocodazole. (B) Bub3-depleted cells exhibit abnormal

    Article Snippet: Unless otherwise stated, incubation with MG132 or with ZM447439 was for 1 h. To evaluate mitotic arrest, cells were incubated with the reversible microtubule depolymerizing drug nocodazole (1 μM, Sigma) for 16 h. Mitotic index was determined by cell-rounding under phase-contrast microscopy.

    Techniques: Transfection

    TUBA1B and TUBB3 are the interacting proteins of CLDN11; CLDN11 blocks cell migration by interfering with tubulin polymerization. a TW02 cells transfected with FLAG-tagged CLDN11 construct (C) or vector control plasmid (V) were harvested after 48 h. TW02 cell extracts and M2 beads were used for a co-immunoprecipitation assay. CLDN11-interacting proteins identified through LC–MS/MS and top-ranking proteins are listed in the table. b Immunoblot analyses were performed to confirm the interaction between FLAG-tagged CLDN11 and endogenous TUBA1B (left panel) or TUBB3 (right panel). c Subcellular distribution of exogenous FLAG-tagged CLDN11 and endogenous TUBA1B or TUBB3 in TW02 cells was assayed through immunofluorescence staining 24 h after transfection. The fluorescent signal (pixel) of each individual cell was quantitated by using the Zen 2.0 software ( n = 50). The proportion of colocalized fluorescent signals (%) is indicated (FLAG-tagged CLDN11, green; Tubulins, red; DAPI, blue). d Schematic illustrations of WT and four deletion FLAG-tagged CLDN11 molecules—transmembrane (TM), extracellular loop (ECL), intracellular loop (ICL), and C-terminus (C). Various FLAG-tagged CLDN11 deletion clones or a vector control were used to dissect the interacting domains on CLDN11 that are crucial for the interaction of endogenous TUBA1B and TUBB3. Input (3%) and immunoprecipitates (30% IP) were assayed through immunoblot analysis by using appropriate antibodies (anti-Flag, anti-TUBA1B and anti-TUBB3). The asterisks denote the major bands of ectopic CLDN11 in the immunoblot assays. e CLDN11 deletion clones were used to perform cell migration assay and to determine which domains on CLDN11 are necessary for blocking cell migration in TW02 cells. f Cell migration and ( g ) cell viability assays were performed in the presence or absence of nocodazole in TW02 or HK1 cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Inactivation of the tight junction gene CLDN11 by aberrant hypermethylation modulates tubulins polymerization and promotes cell migration in nasopharyngeal carcinoma

    doi: 10.1186/s13046-018-0754-y

    Figure Lengend Snippet: TUBA1B and TUBB3 are the interacting proteins of CLDN11; CLDN11 blocks cell migration by interfering with tubulin polymerization. a TW02 cells transfected with FLAG-tagged CLDN11 construct (C) or vector control plasmid (V) were harvested after 48 h. TW02 cell extracts and M2 beads were used for a co-immunoprecipitation assay. CLDN11-interacting proteins identified through LC–MS/MS and top-ranking proteins are listed in the table. b Immunoblot analyses were performed to confirm the interaction between FLAG-tagged CLDN11 and endogenous TUBA1B (left panel) or TUBB3 (right panel). c Subcellular distribution of exogenous FLAG-tagged CLDN11 and endogenous TUBA1B or TUBB3 in TW02 cells was assayed through immunofluorescence staining 24 h after transfection. The fluorescent signal (pixel) of each individual cell was quantitated by using the Zen 2.0 software ( n = 50). The proportion of colocalized fluorescent signals (%) is indicated (FLAG-tagged CLDN11, green; Tubulins, red; DAPI, blue). d Schematic illustrations of WT and four deletion FLAG-tagged CLDN11 molecules—transmembrane (TM), extracellular loop (ECL), intracellular loop (ICL), and C-terminus (C). Various FLAG-tagged CLDN11 deletion clones or a vector control were used to dissect the interacting domains on CLDN11 that are crucial for the interaction of endogenous TUBA1B and TUBB3. Input (3%) and immunoprecipitates (30% IP) were assayed through immunoblot analysis by using appropriate antibodies (anti-Flag, anti-TUBA1B and anti-TUBB3). The asterisks denote the major bands of ectopic CLDN11 in the immunoblot assays. e CLDN11 deletion clones were used to perform cell migration assay and to determine which domains on CLDN11 are necessary for blocking cell migration in TW02 cells. f Cell migration and ( g ) cell viability assays were performed in the presence or absence of nocodazole in TW02 or HK1 cells

    Article Snippet: TW02 or HK1 cells were treated with or without tubulin polymerization inhibitor nocodazole (Sigma).

    Techniques: Migration, Transfection, Construct, Plasmid Preparation, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Immunofluorescence, Staining, Software, Clone Assay, Cell Migration Assay, Blocking Assay

    A Model for transcriptional silencing of CLDN11 through hypermethylation promotes migration by derepression of tubulin polymerization. In a normal nasopharynx, CLDN11 is transcriptionally activated by transcription activators, GATA1 and GATA2. The integral membrane tight junction protein CLDN11, expressed on the apical surface of the epithelial cells, maintains tight junction integrity and epithelial cell polarity and morphology. In addition, CLDN11 serves as the scaffold to recruit tubulins through its intracellular loop and C-terminal domains. The interaction between CLDN11 and the tubulins TUBA1B and TUBB3 may sequester the availability of α- and β-tubulin subunits in the cytoplasm. Thus, the presence of CLDN11 may prevent cell migration and invasion by interfering with the microtubule polymerization dynamics. By contrast, in NPC cells, aberrant promoter hypermethylation impairs GATA binding and causes transcriptional silencing of CLDN11 . In the absence of CLDN11, microtubules undergo rapid polymerization, in turn promoting basement membrane breakdown, motility, invasiveness, plasticity, and cell cycle, thus contributing to a more cancerous phenotype of NPC cells. The tubulin polymerization inhibitor nocodazole can serve as a therapeutic drug to block migration in NPC

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Inactivation of the tight junction gene CLDN11 by aberrant hypermethylation modulates tubulins polymerization and promotes cell migration in nasopharyngeal carcinoma

    doi: 10.1186/s13046-018-0754-y

    Figure Lengend Snippet: A Model for transcriptional silencing of CLDN11 through hypermethylation promotes migration by derepression of tubulin polymerization. In a normal nasopharynx, CLDN11 is transcriptionally activated by transcription activators, GATA1 and GATA2. The integral membrane tight junction protein CLDN11, expressed on the apical surface of the epithelial cells, maintains tight junction integrity and epithelial cell polarity and morphology. In addition, CLDN11 serves as the scaffold to recruit tubulins through its intracellular loop and C-terminal domains. The interaction between CLDN11 and the tubulins TUBA1B and TUBB3 may sequester the availability of α- and β-tubulin subunits in the cytoplasm. Thus, the presence of CLDN11 may prevent cell migration and invasion by interfering with the microtubule polymerization dynamics. By contrast, in NPC cells, aberrant promoter hypermethylation impairs GATA binding and causes transcriptional silencing of CLDN11 . In the absence of CLDN11, microtubules undergo rapid polymerization, in turn promoting basement membrane breakdown, motility, invasiveness, plasticity, and cell cycle, thus contributing to a more cancerous phenotype of NPC cells. The tubulin polymerization inhibitor nocodazole can serve as a therapeutic drug to block migration in NPC

    Article Snippet: TW02 or HK1 cells were treated with or without tubulin polymerization inhibitor nocodazole (Sigma).

    Techniques: Migration, Binding Assay, Blocking Assay

    USP15 isoforms are dynamically expressed and differentially phosphorylated during the cell cycle. a , b A549 cells were synchronised using standard thymidine/nocodazole protocols to enrich for the indicated phases. a USP15 transcript levels remain stable during the cell cycle. Expression of USP15 splice variants and cyclin B1 (CCNB1) were quantified by qRT-PCR, mean data from three independent experiments are expressed relative to actin and normalised to the expression in asynchronous cells (As) for each splice variant. b USP15 protein expression levels oscillate through the cell cycle. USP15 was evaluated by immunoblotting; a representative gel is shown with quantification of total USP15 expression relative to actin below (mean of three independent experiments, error bars SD, one-way ANOVA with Tukey’s multiple comparison test * P ≤ 0.05, ** P ≤ 0.01). c – e USP15 isoform-1 increases in abundance by G2/M and becomes phosphorylated. A549 cells were depleted of USP15 isoforms as indicated and protein extracts were compared by immunoblotting for asynchronous cells (As) or cells arrested at G2/M. Separation by 4–12% gradient SDS-PAGE c showing quantification of the siCON2 samples d , and analysis by Phos-tag gel electrophoresis e

    Journal: Oncogene

    Article Title: The deubiquitylase USP15 regulates topoisomerase II alpha to maintain genome integrity

    doi: 10.1038/s41388-017-0092-0

    Figure Lengend Snippet: USP15 isoforms are dynamically expressed and differentially phosphorylated during the cell cycle. a , b A549 cells were synchronised using standard thymidine/nocodazole protocols to enrich for the indicated phases. a USP15 transcript levels remain stable during the cell cycle. Expression of USP15 splice variants and cyclin B1 (CCNB1) were quantified by qRT-PCR, mean data from three independent experiments are expressed relative to actin and normalised to the expression in asynchronous cells (As) for each splice variant. b USP15 protein expression levels oscillate through the cell cycle. USP15 was evaluated by immunoblotting; a representative gel is shown with quantification of total USP15 expression relative to actin below (mean of three independent experiments, error bars SD, one-way ANOVA with Tukey’s multiple comparison test * P ≤ 0.05, ** P ≤ 0.01). c – e USP15 isoform-1 increases in abundance by G2/M and becomes phosphorylated. A549 cells were depleted of USP15 isoforms as indicated and protein extracts were compared by immunoblotting for asynchronous cells (As) or cells arrested at G2/M. Separation by 4–12% gradient SDS-PAGE c showing quantification of the siCON2 samples d , and analysis by Phos-tag gel electrophoresis e

    Article Snippet: To obtain cell populations synchronised at G1/S or in early S, late S, or G2, A549 were subject to double-thymidine block (2 mM thymidine (Sigma) for 18 h, released into fresh media for 8 h, arrested with thymidine for 17 h) then lysed directly or released into full medium for 2.5, 5.5, or 7.5 h. To obtain cell populations synchronised at G2/M or in M, or early G1, A549 were subject to a thymidine/nocodazole block (2 mM thymidine for 24 h, released into full medium containing 100ng/ml nocodazole (Sigma) for 14 h) then mitotic cells were collected by knocking off the dish, and either lysed directly or replated into full medium for 0.5 or 4 h. Propidium iodide staining with flow cytometry analysis, and immunoblotting for a panel of stage-specific cell cycle markers, are routinely used to confirm enrichment of cell cycle phases.

    Techniques: Expressing, Quantitative RT-PCR, Variant Assay, SDS Page, Nucleic Acid Electrophoresis

    A, Microtubule disruption abrogates the inhibitory effect of cGMP on TGF-β-induced PAI-1 mRNA expression. Serum-starved PASMC were pretreated with nocodazole (5 μg/ml) or vehicle (control) for 1 h, followed by cGMP (0.5 m m ) for 1 h, and

    Journal: Molecular Endocrinology

    Article Title: cGMP Inhibits TGF-? Signaling by Sequestering Smad3 with Cytosolic ?2-Tubulin in Pulmonary Artery Smooth Muscle Cells

    doi: 10.1210/me.2011-1009

    Figure Lengend Snippet: A, Microtubule disruption abrogates the inhibitory effect of cGMP on TGF-β-induced PAI-1 mRNA expression. Serum-starved PASMC were pretreated with nocodazole (5 μg/ml) or vehicle (control) for 1 h, followed by cGMP (0.5 m m ) for 1 h, and

    Article Snippet: To determine the effects of cGMP, nocodazole/colchicine (catalog no. C9754; Sigma-Aldrich) and paclitaxel/epothilone B (catalog no.E2656; Sigma-Aldrich) on TGF-β1-induced PAI-1 mRNA expression, quiescent PASMC were pretreated with nocodazole (5 μg/ml)/colchicine (1 μ m ), paclitaxel (1 μ m )/epothilone B (50 ng/ml), or vehicle for 1 h, followed by cGMP (0.5 m m ) for 1 h and then exposed to TGF-β1 (2 ng/ml) for additional 12 h. Total RNA was extracted using TRIzol Reagent (catalog no. 15596-018; Invitrogen).

    Techniques: Expressing