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    Structured Review

    Millipore nocodazole
    (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) <t>Nocodazole</t> 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.
    Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae"

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

    Journal: Molecular Biology of the Cell

    doi:

    (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.
    Figure Legend 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.

    Techniques Used: Expressing, Fluorescence, Microscopy, Incubation, Staining

    2) Product Images from "APC/CCdc20 targets E2F1 for degradation in prometaphase"

    Article Title: APC/CCdc20 targets E2F1 for degradation in prometaphase

    Journal: Cell Cycle

    doi: 10.4161/cc.9.19.13162

    Cdc20 targets E2F1 for degradation in prometaphase. (A) HeLa cells were transfected with control (Luc), Cdc20 or Cdh1 siRNA oligos. 32 h later, fresh medium was added and cells were treated with 330 nM nocodazole for an additional 16 h to arrest cells in prometaphase. Cells were then replated in fresh media and harvested at the indicated times after release. Extracts were prepared and analyzed by immunoblotting with antibodies to the indicated proteins. (B) HeLa cells were transfected with control (Luc) or Cdc20 siRNA oligos. 32 h later, fresh medium was added and cells were left untreated (asynchronous) or treated with 330 nM nocodazole (Noc) for an additional 16 h to arrest cells in prometaphase. Total RNA was isolated and quantitative RT-PCR analysis was performed. Relative E2F1 mRNA levels were calculated by normalizing to L32 and data are representative of three independent experiments.
    Figure Legend Snippet: Cdc20 targets E2F1 for degradation in prometaphase. (A) HeLa cells were transfected with control (Luc), Cdc20 or Cdh1 siRNA oligos. 32 h later, fresh medium was added and cells were treated with 330 nM nocodazole for an additional 16 h to arrest cells in prometaphase. Cells were then replated in fresh media and harvested at the indicated times after release. Extracts were prepared and analyzed by immunoblotting with antibodies to the indicated proteins. (B) HeLa cells were transfected with control (Luc) or Cdc20 siRNA oligos. 32 h later, fresh medium was added and cells were left untreated (asynchronous) or treated with 330 nM nocodazole (Noc) for an additional 16 h to arrest cells in prometaphase. Total RNA was isolated and quantitative RT-PCR analysis was performed. Relative E2F1 mRNA levels were calculated by normalizing to L32 and data are representative of three independent experiments.

    Techniques Used: Transfection, Isolation, Quantitative RT-PCR

    E2F-1 is degraded in late S/G 2 phase. (A and B) HeLa cells were synchronized in prometaphase with 330 nM nocodazole for 16 h, collected and replated in fresh medium. Cells were harvested at the indicated time points and processed for immunoblotting (A) or FACS analysis (B). (C and D) HeLa cells were synchronized at G 1 /S using a double thymidine block as described in Materials and Methods. Following release from the double thymidine block, cells were harvested at the indicated time points and processed for immunoblotting (C) or FACS analysis (D).
    Figure Legend Snippet: E2F-1 is degraded in late S/G 2 phase. (A and B) HeLa cells were synchronized in prometaphase with 330 nM nocodazole for 16 h, collected and replated in fresh medium. Cells were harvested at the indicated time points and processed for immunoblotting (A) or FACS analysis (B). (C and D) HeLa cells were synchronized at G 1 /S using a double thymidine block as described in Materials and Methods. Following release from the double thymidine block, cells were harvested at the indicated time points and processed for immunoblotting (C) or FACS analysis (D).

    Techniques Used: FACS, Blocking Assay

    3) Product Images from "Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1"

    Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06854-2

    Expression of ASXL2 K370R reduces mammalian cell proliferation. a Enforced expression of ASXL2 K370R decreases cellular proliferation. U-2 OS cells were transduced with different amounts of lentiviral suspensions produced using ASXL2 or ASXL2 K370R constructs. Cells were selected by puromycin and harvested for immunoblotting (top panel). Equal numbers of puromycin-selected cells were plated for colony formation assay (CFA) (bottom panel). n = 2 biological replicates. b The cells infected in a , were treated with nocodazole for FACS analysis at the indicated times. Note that (+2×) refers to transduction of cells with twice the amount of virus we normally use for Myc-ASXL2, and (−2×) refers to transduction of the cells with two times less the amount of viruses we normally use for ASXL2 370R. This adjustment was conducted to correct for the expression levels usually higher for ASXL2 K370R. n = 2 biological replicates. c , d Normal diploid fibroblast IMR90 cells were transduced with viral expression constructs for ASXL2 or ASXL2 K370R. Cells were selected by puromycin and equal numbers were plated for phase contrast pictures ( c ) or cell counts ( d ). Scale bar: 50 µm for panel c . n = 2 biological replicates. (Exp.1 and Exp.2). e siRNA depletion of ASXL2 decreases cellular proliferation. U-2 OS cells were transfected with NT siRNA control or siRNA for ASXL2 . Equal numbers of puromycin-selected cells were plated for CFA (left panel). Cells were treated with nocodazole for FACS analysis (right panel). f siRNA depletion of UBE2E3 decreases cellular proliferation. U-2 OS cells were transfected with individual siRNA constructs as indicated. Cells were plated for viability measurement using MTT assay. n = 3 biological replicates. Error bars represent s.d. (mean ± SD). g , h Inactivation of UBE2E3 locus decreases cellular proliferation. Schematic representation for gRNAs targeting the UBE2E3 locus ( g top panel). U-2 OS cells were transduced with different lentiviral CRISPR/Cas9 constructs, selected by puromycin and harvested for immunoblotting ( g bottom panel). n = 3 biological replicates. Equal numbers of puromycin-selected cells were plated for CFA ( h ). n = 2 biological replicates. i The cells selected as in h were treated with nocodazole for FACS analysis at the indicated time . n = 2 biological replicates. j ). Pictures were taken at 100× magnification. Scale bar: 100 μm. Tubulin was used as a loading control for panels a and g
    Figure Legend Snippet: Expression of ASXL2 K370R reduces mammalian cell proliferation. a Enforced expression of ASXL2 K370R decreases cellular proliferation. U-2 OS cells were transduced with different amounts of lentiviral suspensions produced using ASXL2 or ASXL2 K370R constructs. Cells were selected by puromycin and harvested for immunoblotting (top panel). Equal numbers of puromycin-selected cells were plated for colony formation assay (CFA) (bottom panel). n = 2 biological replicates. b The cells infected in a , were treated with nocodazole for FACS analysis at the indicated times. Note that (+2×) refers to transduction of cells with twice the amount of virus we normally use for Myc-ASXL2, and (−2×) refers to transduction of the cells with two times less the amount of viruses we normally use for ASXL2 370R. This adjustment was conducted to correct for the expression levels usually higher for ASXL2 K370R. n = 2 biological replicates. c , d Normal diploid fibroblast IMR90 cells were transduced with viral expression constructs for ASXL2 or ASXL2 K370R. Cells were selected by puromycin and equal numbers were plated for phase contrast pictures ( c ) or cell counts ( d ). Scale bar: 50 µm for panel c . n = 2 biological replicates. (Exp.1 and Exp.2). e siRNA depletion of ASXL2 decreases cellular proliferation. U-2 OS cells were transfected with NT siRNA control or siRNA for ASXL2 . Equal numbers of puromycin-selected cells were plated for CFA (left panel). Cells were treated with nocodazole for FACS analysis (right panel). f siRNA depletion of UBE2E3 decreases cellular proliferation. U-2 OS cells were transfected with individual siRNA constructs as indicated. Cells were plated for viability measurement using MTT assay. n = 3 biological replicates. Error bars represent s.d. (mean ± SD). g , h Inactivation of UBE2E3 locus decreases cellular proliferation. Schematic representation for gRNAs targeting the UBE2E3 locus ( g top panel). U-2 OS cells were transduced with different lentiviral CRISPR/Cas9 constructs, selected by puromycin and harvested for immunoblotting ( g bottom panel). n = 3 biological replicates. Equal numbers of puromycin-selected cells were plated for CFA ( h ). n = 2 biological replicates. i The cells selected as in h were treated with nocodazole for FACS analysis at the indicated time . n = 2 biological replicates. j ). Pictures were taken at 100× magnification. Scale bar: 100 μm. Tubulin was used as a loading control for panels a and g

    Techniques Used: Expressing, Transduction, Produced, Construct, Colony Assay, Infection, FACS, Transfection, MTT Assay, CRISPR

    4) Product Images from "Elm1 kinase activates the spindle position checkpoint kinase Kin4"

    Article Title: Elm1 kinase activates the spindle position checkpoint kinase Kin4

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201006151

    Bud neck localization of Elm1 is not necessary for Kin4 activity. (A) Still images of live KIN4-GFP ELM1-3mCherry cells. Bars, 3 µm. (B) Radioactive kinase assay of immunoprecipitated Kin4-6HA from Gal1 -clb2ΔDB –overexpressing cells arrested with nocodazole with a bud neck (lane 1) and without a bud neck (lanes 2 and 3). The percentages of budded and nonbudded cells are indicated. See Materials and methods for details. (C) Both Gal1 -clb2ΔDB and Gal1 -clb2ΔDB elm1Δ cells were arrested with α-factor (G1-phase, t 0 ) and forced to enter mitosis without bud formation upon clb2ΔDB overexpression in the presence of nocodazole. Samples were taken every hour and analyzed by immunoblotting. The asterisk marks Kin4-hyperphosphorylated forms. (D) cdc12-6 and cdc12-6 elm1Δ cells were arrested at 23°C with α-factor and released at 37°C in nocodazole-containing medium. In vitro kinase assays were performed using immunoprecipitated Kin4-6HA (anti-HA blot) and MBP-Bfa1 (Coomassie). Incorporation of the 32 P isotope in MBP-Bfa1 was measured by autoradiography.
    Figure Legend Snippet: Bud neck localization of Elm1 is not necessary for Kin4 activity. (A) Still images of live KIN4-GFP ELM1-3mCherry cells. Bars, 3 µm. (B) Radioactive kinase assay of immunoprecipitated Kin4-6HA from Gal1 -clb2ΔDB –overexpressing cells arrested with nocodazole with a bud neck (lane 1) and without a bud neck (lanes 2 and 3). The percentages of budded and nonbudded cells are indicated. See Materials and methods for details. (C) Both Gal1 -clb2ΔDB and Gal1 -clb2ΔDB elm1Δ cells were arrested with α-factor (G1-phase, t 0 ) and forced to enter mitosis without bud formation upon clb2ΔDB overexpression in the presence of nocodazole. Samples were taken every hour and analyzed by immunoblotting. The asterisk marks Kin4-hyperphosphorylated forms. (D) cdc12-6 and cdc12-6 elm1Δ cells were arrested at 23°C with α-factor and released at 37°C in nocodazole-containing medium. In vitro kinase assays were performed using immunoprecipitated Kin4-6HA (anti-HA blot) and MBP-Bfa1 (Coomassie). Incorporation of the 32 P isotope in MBP-Bfa1 was measured by autoradiography.

    Techniques Used: Activity Assay, Kinase Assay, Immunoprecipitation, Over Expression, In Vitro, Autoradiography

    Bfa1 localization and phosphorylation in elm1Δ and rts1Δ cells. (A–C) Met3- CDC20 Gal1- KIN4 SPC42-eqFP mCherry-TUB1 cells were arrested in metaphase by Cdc20 depletion followed by the addition of galactose (GAL, induction of Gal1- KIN4 ) or glucose (GLU, repression of Gal1- KIN4 ) for 3 h. Spc42 served as an SPB marker. Localization of Bfa1-GFP at SPBs was inspected after fixing the cells with paraformaldehyde. Bars, 3 µm. (D) Quantification of A–C. Bfa1-GFP was considered symmetric if equally bound to both SPBs and asymmetric if strongly bound to one of the two SPBs. (E) The indicated strains were arrested at G1 phase with α-factor and released in nocodazole-containing media. Bfa1 and Clb2 levels were determined by immunoblotting at the indicated times. Tub1 served as loading control. Asterisks indicate hyperphosphorylated Bfa1 forms. The percentage of large- and multibudded cells was plotted versus time.
    Figure Legend Snippet: Bfa1 localization and phosphorylation in elm1Δ and rts1Δ cells. (A–C) Met3- CDC20 Gal1- KIN4 SPC42-eqFP mCherry-TUB1 cells were arrested in metaphase by Cdc20 depletion followed by the addition of galactose (GAL, induction of Gal1- KIN4 ) or glucose (GLU, repression of Gal1- KIN4 ) for 3 h. Spc42 served as an SPB marker. Localization of Bfa1-GFP at SPBs was inspected after fixing the cells with paraformaldehyde. Bars, 3 µm. (D) Quantification of A–C. Bfa1-GFP was considered symmetric if equally bound to both SPBs and asymmetric if strongly bound to one of the two SPBs. (E) The indicated strains were arrested at G1 phase with α-factor and released in nocodazole-containing media. Bfa1 and Clb2 levels were determined by immunoblotting at the indicated times. Tub1 served as loading control. Asterisks indicate hyperphosphorylated Bfa1 forms. The percentage of large- and multibudded cells was plotted versus time.

    Techniques Used: Marker

    Elm1 phosphorylates Kin4 at threonine 209. (A) Indicated cells types were arrested in the G1 phase by α-factor (t 0 ) and released in nocodazole-containing medium. Samples were taken every hour and probed for Kin4-6HA, Clb2, and Sic1 by immunoblotting. H + ATPase served as loading control. (B) Sequence alignment of Kin4, Snf1, and Hsl1 activation loops. Identical residues are shown in black. The conserved threonine residue is enlarged. (C) α-Factor–arrested cells were released in nocodazole-containing medium. Samples were collected every hour and tested by immunoblotting as in A. (D) Cycling cultures of strains carrying KIN4 (no tag), KIN4-6HA (WT), kin4-T209A-6HA (T209A), and KIN4-6HA elm1Δ were subjected to immunoprecipitation using anti-HA beads. Samples were probed with anti-HA and anti–T209-P antibodies by immunoblotting. The brackets depict the slower- and faster-migrating Kin4 forms. (E) In vitro phosphorylated GST-Kin4 and GST-Kin4-T209A by GST-Elm1ΔC were probed with anti-T209-P and anti-GST antibodies. Asterisks in A and C indicate hyperphosphorylated forms of Kin4.
    Figure Legend Snippet: Elm1 phosphorylates Kin4 at threonine 209. (A) Indicated cells types were arrested in the G1 phase by α-factor (t 0 ) and released in nocodazole-containing medium. Samples were taken every hour and probed for Kin4-6HA, Clb2, and Sic1 by immunoblotting. H + ATPase served as loading control. (B) Sequence alignment of Kin4, Snf1, and Hsl1 activation loops. Identical residues are shown in black. The conserved threonine residue is enlarged. (C) α-Factor–arrested cells were released in nocodazole-containing medium. Samples were collected every hour and tested by immunoblotting as in A. (D) Cycling cultures of strains carrying KIN4 (no tag), KIN4-6HA (WT), kin4-T209A-6HA (T209A), and KIN4-6HA elm1Δ were subjected to immunoprecipitation using anti-HA beads. Samples were probed with anti-HA and anti–T209-P antibodies by immunoblotting. The brackets depict the slower- and faster-migrating Kin4 forms. (E) In vitro phosphorylated GST-Kin4 and GST-Kin4-T209A by GST-Elm1ΔC were probed with anti-T209-P and anti-GST antibodies. Asterisks in A and C indicate hyperphosphorylated forms of Kin4.

    Techniques Used: Sequencing, Activation Assay, Immunoprecipitation, In Vitro

    Localization and activity of Kin4 in elm1Δ cells. (A) Kin4-GFP localization in nocodazole-arrested cells. Spc42-eqFP served as an SPB marker. Images were taken without fixation. Note that the two SPBs collapse, forming a large single eqFP signal, due to depolymerization of the microtubules by nocodazole. Bars, 3 µm. (B) Quantification of A showing one representative experiment out of three; 100–150 cells were counted per sample. (C) In vitro kinase assay using immunoprecipitated Kin4-6HA (lanes 2, 3, 5, and 6) or Kin4-T209A-6HA (lane 4) from cycling cultures of wild type (WT), rts1Δ , or elm1Δ cells as indicated. Nontagged Kin4 (lane 1) was used as a control for contaminating kinases. Kin4-6HA was detected by immunoblotting (anti-HA). MBP-Bfa1 was used as a substrate (Coomassie staining). Incorporation of the 32 P isotope was determined by autoradiography. Note that a 66-kD degradation product of MBP-Bfa1 was phosphorylated efficiently by Kin4-6HA as described in Maekawa et al. (2007) . Plus and minus represent the presence and the absence of MBP-Bfa1. Kin4 did not phosphorylate MBP alone ( Maekawa et al., 2007 ; not depicted).
    Figure Legend Snippet: Localization and activity of Kin4 in elm1Δ cells. (A) Kin4-GFP localization in nocodazole-arrested cells. Spc42-eqFP served as an SPB marker. Images were taken without fixation. Note that the two SPBs collapse, forming a large single eqFP signal, due to depolymerization of the microtubules by nocodazole. Bars, 3 µm. (B) Quantification of A showing one representative experiment out of three; 100–150 cells were counted per sample. (C) In vitro kinase assay using immunoprecipitated Kin4-6HA (lanes 2, 3, 5, and 6) or Kin4-T209A-6HA (lane 4) from cycling cultures of wild type (WT), rts1Δ , or elm1Δ cells as indicated. Nontagged Kin4 (lane 1) was used as a control for contaminating kinases. Kin4-6HA was detected by immunoblotting (anti-HA). MBP-Bfa1 was used as a substrate (Coomassie staining). Incorporation of the 32 P isotope was determined by autoradiography. Note that a 66-kD degradation product of MBP-Bfa1 was phosphorylated efficiently by Kin4-6HA as described in Maekawa et al. (2007) . Plus and minus represent the presence and the absence of MBP-Bfa1. Kin4 did not phosphorylate MBP alone ( Maekawa et al., 2007 ; not depicted).

    Techniques Used: Activity Assay, Marker, In Vitro, Kinase Assay, Immunoprecipitation, Staining, Autoradiography

    5) Product Images from "Mammalian Cdh1/Fzr mediates its own degradation"

    Article Title: Mammalian Cdh1/Fzr mediates its own degradation

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600149

    Cdh1 is degraded in G1. Cells were synchronized with nocodazole and prometaphase-arrested cells were obtained by mitotic shake-off. Cells were released into fresh medium and after 1 h cells were either treated (+) or not treated (−) with 100 μg/ml cyclohexamide. Cells were harvested and immunoblotted at the indicated time points with Cdh1 and actin antibodies. The histogram shows the quantitative data from three different experiments.
    Figure Legend Snippet: Cdh1 is degraded in G1. Cells were synchronized with nocodazole and prometaphase-arrested cells were obtained by mitotic shake-off. Cells were released into fresh medium and after 1 h cells were either treated (+) or not treated (−) with 100 μg/ml cyclohexamide. Cells were harvested and immunoblotted at the indicated time points with Cdh1 and actin antibodies. The histogram shows the quantitative data from three different experiments.

    Techniques Used:

    During G1 and G0, most of the Cdh1 in the cell is associated with the APC/C. ( A ) Extracts of nocodazole (noc)-arrested G1 prepared by releasing nocodazole-arrested cells for 6 h and G0 cells prepared by 48 h serum deprivation were fractionated by gel filtration. Samples of each fraction were blotted with Cdc27 and Cdh1 antibodies. ( B ) Fraction 4 of gel filtrates of cell extracts of the different phases were resolved side by side to show that Cdc27 was phosphorylated in prometaphase, but not in G1 or G0.
    Figure Legend Snippet: During G1 and G0, most of the Cdh1 in the cell is associated with the APC/C. ( A ) Extracts of nocodazole (noc)-arrested G1 prepared by releasing nocodazole-arrested cells for 6 h and G0 cells prepared by 48 h serum deprivation were fractionated by gel filtration. Samples of each fraction were blotted with Cdc27 and Cdh1 antibodies. ( B ) Fraction 4 of gel filtrates of cell extracts of the different phases were resolved side by side to show that Cdc27 was phosphorylated in prometaphase, but not in G1 or G0.

    Techniques Used: Filtration

    Cdh1 has two RXXL boxes that are required for its degradation in G1. ( A ) Schematic presentation of the fusion constructs of wild-type and RXXL box mutants, as well as an N-terminally myc-tagged Cdh-CAT construct. The numbers above depict the location of the amino acid in the protein; the constructs are not drawn to scale. ( B ) Cells stably expressing the various fusion proteins shown in (A), as well as cells expressing CAT, were arrested with nocodazole and obtained by mitotic shake-off. Cells were either harvested immediately (black) or released into fresh medium and harvested after 3 (early G1—gray) or 6 (mid-late G1—white) h for CAT assays. ( C ) Full-length Cdh1, Cdh1-DM1 and Cdh1-DM2 were transcribed and translated in reticulocyte lysate in the presence of [ 35 S]methionine. They were subsequently incubated with APC/C purified from interphase cells, E2-C, E1, ubiquitin and an energy-regenerating system, and resolved by SDS–PAGE. ( D ) Cells were injected with expression vectors for Cdh-YFP (top) and Cdh-DM1+2-YFP (bottom). Expressing cells were followed by time-lapse photography and photographed every 5 min. Representative images showing the same cells 1 h before metaphase (−1 h) at metaphase (0 h), and every 2 h for 8 h after metaphase are presented.
    Figure Legend Snippet: Cdh1 has two RXXL boxes that are required for its degradation in G1. ( A ) Schematic presentation of the fusion constructs of wild-type and RXXL box mutants, as well as an N-terminally myc-tagged Cdh-CAT construct. The numbers above depict the location of the amino acid in the protein; the constructs are not drawn to scale. ( B ) Cells stably expressing the various fusion proteins shown in (A), as well as cells expressing CAT, were arrested with nocodazole and obtained by mitotic shake-off. Cells were either harvested immediately (black) or released into fresh medium and harvested after 3 (early G1—gray) or 6 (mid-late G1—white) h for CAT assays. ( C ) Full-length Cdh1, Cdh1-DM1 and Cdh1-DM2 were transcribed and translated in reticulocyte lysate in the presence of [ 35 S]methionine. They were subsequently incubated with APC/C purified from interphase cells, E2-C, E1, ubiquitin and an energy-regenerating system, and resolved by SDS–PAGE. ( D ) Cells were injected with expression vectors for Cdh-YFP (top) and Cdh-DM1+2-YFP (bottom). Expressing cells were followed by time-lapse photography and photographed every 5 min. Representative images showing the same cells 1 h before metaphase (−1 h) at metaphase (0 h), and every 2 h for 8 h after metaphase are presented.

    Techniques Used: Construct, Stable Transfection, Expressing, Incubation, Purification, SDS Page, Injection

    Cdh-CAT fusion proteins are degraded in G1 and G0. ( A ) A vector expressing a fusion between the N-terminal 180 amino acids of Cdh1 and CAT, and a vector expressing CAT alone, were stably expressed in cells. Cells were synchronized by nocodazole arrest and mitotic shake-off, released into fresh medium, harvested at the indicated times and assayed for CAT activity. The plotted CAT activity represents the proportion of di-acetylated [ 14 C]chloramphenicol of the total amount of [ 14 C]chloramphenicol. ( B ) The same cell lines were arrested in G0 by serum deprivation and released by addition of serum. Cells were harvested at the indicated times and assayed for CAT activity.
    Figure Legend Snippet: Cdh-CAT fusion proteins are degraded in G1 and G0. ( A ) A vector expressing a fusion between the N-terminal 180 amino acids of Cdh1 and CAT, and a vector expressing CAT alone, were stably expressed in cells. Cells were synchronized by nocodazole arrest and mitotic shake-off, released into fresh medium, harvested at the indicated times and assayed for CAT activity. The plotted CAT activity represents the proportion of di-acetylated [ 14 C]chloramphenicol of the total amount of [ 14 C]chloramphenicol. ( B ) The same cell lines were arrested in G0 by serum deprivation and released by addition of serum. Cells were harvested at the indicated times and assayed for CAT activity.

    Techniques Used: Plasmid Preparation, Expressing, Stable Transfection, Activity Assay

    Cdh1 degradation is mediated by the APC/C Cdh1 and not by the APC/C Cdc20 . ( A ) Cells stably expressing Cdh-CAT, Cdh-DM1-CAT and cyclin B1-CAT were synchronized in prometaphase with nocodazole (black), in telophase by expression of nondegradable cyclin B1 (gray) and in G1 by release from nocodazole arrest (white), and assayed for CAT activity. ( B ) Cells were transiently co-transfected with a full-length Cdh1 expression vector (+) or an empty vector (−), and with Cdh-CAT, Cdh-DM1-CAT or cyclin B1-CAT as indicated. The transfected cells were arrested with nocodazole, harvested by shake-off and assayed for CAT activity. ( C ) Cells stably expressing Cdh-CAT, cyclin B1-CAT and CAT were arrested with nocodazole overnight and harvested by shake-off. They were then incubated in medium with both nocodazole and roscovitin for 3 h, harvested and assayed for CAT activity. A representative experiment of at least three repeats is shown for each of the experiments.
    Figure Legend Snippet: Cdh1 degradation is mediated by the APC/C Cdh1 and not by the APC/C Cdc20 . ( A ) Cells stably expressing Cdh-CAT, Cdh-DM1-CAT and cyclin B1-CAT were synchronized in prometaphase with nocodazole (black), in telophase by expression of nondegradable cyclin B1 (gray) and in G1 by release from nocodazole arrest (white), and assayed for CAT activity. ( B ) Cells were transiently co-transfected with a full-length Cdh1 expression vector (+) or an empty vector (−), and with Cdh-CAT, Cdh-DM1-CAT or cyclin B1-CAT as indicated. The transfected cells were arrested with nocodazole, harvested by shake-off and assayed for CAT activity. ( C ) Cells stably expressing Cdh-CAT, cyclin B1-CAT and CAT were arrested with nocodazole overnight and harvested by shake-off. They were then incubated in medium with both nocodazole and roscovitin for 3 h, harvested and assayed for CAT activity. A representative experiment of at least three repeats is shown for each of the experiments.

    Techniques Used: Stable Transfection, Expressing, Activity Assay, Transfection, Plasmid Preparation, Incubation

    Levels of Cdh1 oscillate during the cell cycle. ( A ) Cells synchronized in prometaphase by nocodazole arrest and shake-off were released and harvested at the indicated time points. Cell extracts were immunoblotted with Cdh1, cyclin B1 and actin antibodies. ( B ) Cells were synchronized by serum deprivation in G0, released and harvested at the indicated times for immunoblotting with Cdh1 and actin antibodies. ( C ) The ratio between Cdh1 and actin in nocodazole-arrested, mid-G1 (6 h after release from nocodazole) and serum-starved (G0) cells was calculated from Cdh1 and actin signals quantified on a FUJIFILM LAS-1000 intelligent dark box II. The amount of Cdh1 in nocodazole-arrested cells was set arbitrarily to 100%.
    Figure Legend Snippet: Levels of Cdh1 oscillate during the cell cycle. ( A ) Cells synchronized in prometaphase by nocodazole arrest and shake-off were released and harvested at the indicated time points. Cell extracts were immunoblotted with Cdh1, cyclin B1 and actin antibodies. ( B ) Cells were synchronized by serum deprivation in G0, released and harvested at the indicated times for immunoblotting with Cdh1 and actin antibodies. ( C ) The ratio between Cdh1 and actin in nocodazole-arrested, mid-G1 (6 h after release from nocodazole) and serum-starved (G0) cells was calculated from Cdh1 and actin signals quantified on a FUJIFILM LAS-1000 intelligent dark box II. The amount of Cdh1 in nocodazole-arrested cells was set arbitrarily to 100%.

    Techniques Used:

    6) Product Images from "Sumoylated BubR1 plays an important role in chromosome segregation and mitotic timing"

    Article Title: Sumoylated BubR1 plays an important role in chromosome segregation and mitotic timing

    Journal: Cell Cycle

    doi: 10.4161/cc.11.4.19307

    BubR1 sumoylation is associated with its biochemical inactivation. (A) Mitotic cells collected by shake-off after treatment with nocodazole for 14 h were released into fresh culture medium. At various times of release, cell lysates, along with interphase
    Figure Legend Snippet: BubR1 sumoylation is associated with its biochemical inactivation. (A) Mitotic cells collected by shake-off after treatment with nocodazole for 14 h were released into fresh culture medium. At various times of release, cell lysates, along with interphase

    Techniques Used:

    Expression of sumoylation-deficient mutant of BubR1 N-terminus induces early anaphase arrest. (A) HeLa cells treated with nocodazole for 14 h. Mitotic cells collected by shaking-off were re-cultured in fresh medium supplemented with or without nocodazole
    Figure Legend Snippet: Expression of sumoylation-deficient mutant of BubR1 N-terminus induces early anaphase arrest. (A) HeLa cells treated with nocodazole for 14 h. Mitotic cells collected by shaking-off were re-cultured in fresh medium supplemented with or without nocodazole

    Techniques Used: Expressing, Mutagenesis, Cell Culture

    BubR1 is modified by sumoylation. (A) HeLa cells were cultured in the presence or absence of nocodazole (Noc, 40 ng/mL) for 18 h. Equal amounts of cell lysates were blotted for BubR1 and β-actin. BubR1 (arrow BubR1), phosphorylated BubR1 (arrow
    Figure Legend Snippet: BubR1 is modified by sumoylation. (A) HeLa cells were cultured in the presence or absence of nocodazole (Noc, 40 ng/mL) for 18 h. Equal amounts of cell lysates were blotted for BubR1 and β-actin. BubR1 (arrow BubR1), phosphorylated BubR1 (arrow

    Techniques Used: Modification, Cell Culture

    7) Product Images from "The Temperature-Sensitive Role of Cryptococcus neoformansROM2 in Cell Morphogenesis"

    Article Title: The Temperature-Sensitive Role of Cryptococcus neoformansROM2 in Cell Morphogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000368

    Sensitivity to nocodazole was assessed using KN99α, KN99α rom2 , and KN99α rom2 + ROM2 . Cultures were grown overnight and serial dilutions were plated on YPD with 2 µM nocodazole. Plates were grown for 2 days at 25°C, 30°C or 37°C to detect growth inhibition.
    Figure Legend Snippet: Sensitivity to nocodazole was assessed using KN99α, KN99α rom2 , and KN99α rom2 + ROM2 . Cultures were grown overnight and serial dilutions were plated on YPD with 2 µM nocodazole. Plates were grown for 2 days at 25°C, 30°C or 37°C to detect growth inhibition.

    Techniques Used: Inhibition

    8) Product Images from "Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy "

    Article Title: Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy

    Journal: The Journal of Cell Biology

    doi:

    Microtubule stability in pressure overload-hypertrophied myocardium: Immunofluorescence confocal micrographs, using the DM1B anti–β-tubulin antibody, of RV and LV cardiocyte microtubules during exposure to nocodazole ( A–H ) or low temperature ( I–L ). Cardiocytes from a feline heart 2 wk after RV pressure overloading were exposed to 0.3 μM nocodazole for 0 min ( A and B ), 30 min ( C and D ), 60 min ( E and F ), or 90 min ( G and H ) or they were exposed to a temperature of 0°C for 0 ( I and J ) or 60 min ( K and L ) before fixation. The cardiocytes on the left ( A, C, E, G, I, and K ) are from the RV, and the cardiocytes on the right ( B, D, F, H, J, and L ) are from the LV. Of interest, after 90 min of nocodazole exposure the majority of the residual microtubules of RV but not LV cardiocytes was found by double-staining to be d ecorated by both the β-tubulin and either the Glu-tubulin or Δ2-tubulin antibodies (data not shown). Each micrograph is a single 0.7-μm confocal section taken at the level of the nuclei. Bar, 25 μm.
    Figure Legend Snippet: Microtubule stability in pressure overload-hypertrophied myocardium: Immunofluorescence confocal micrographs, using the DM1B anti–β-tubulin antibody, of RV and LV cardiocyte microtubules during exposure to nocodazole ( A–H ) or low temperature ( I–L ). Cardiocytes from a feline heart 2 wk after RV pressure overloading were exposed to 0.3 μM nocodazole for 0 min ( A and B ), 30 min ( C and D ), 60 min ( E and F ), or 90 min ( G and H ) or they were exposed to a temperature of 0°C for 0 ( I and J ) or 60 min ( K and L ) before fixation. The cardiocytes on the left ( A, C, E, G, I, and K ) are from the RV, and the cardiocytes on the right ( B, D, F, H, J, and L ) are from the LV. Of interest, after 90 min of nocodazole exposure the majority of the residual microtubules of RV but not LV cardiocytes was found by double-staining to be d ecorated by both the β-tubulin and either the Glu-tubulin or Δ2-tubulin antibodies (data not shown). Each micrograph is a single 0.7-μm confocal section taken at the level of the nuclei. Bar, 25 μm.

    Techniques Used: Immunofluorescence, Double Staining

    9) Product Images from "Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis"

    Article Title: Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis

    Journal: Genes & Development

    doi: 10.1101/gad.622411

    Hof1 phosphomimetic mutations in Dbf2 sites partially rescue dbf2-2 dbf20Δ -dependent cytokinesis defects. Cells of the indicated genotypes were arrested in G1 at 23°C with α-factor. Cells were released in nocodazole at 37°C to promote metaphase arrest and inactivation of Dbf2-2. After release from metaphase, overexpression of SIC1 was induced by addition of galactose to the culture medium to overcome the mitotic exit defects of dbf2-2 dbf20Δ cells. Samples were taken every 30 min to monitor Hof1 localization and actin repolarization. One representative cell (taken from time point 60 min) is shown in A–D . The levels of Hof1-GFP, Sic1, and Clb2 were monitored by immunoblotting using anti-GFP, anti-Sic1, and anti-Clb2 antibodies. Tub1 served as a loading control. The graphs indicate the percentage of cells ( n = 100–150) showing actin repolarization at the bud neck and Hof1-GFP at septins, at the AMR ring, or as a contracted ring.
    Figure Legend Snippet: Hof1 phosphomimetic mutations in Dbf2 sites partially rescue dbf2-2 dbf20Δ -dependent cytokinesis defects. Cells of the indicated genotypes were arrested in G1 at 23°C with α-factor. Cells were released in nocodazole at 37°C to promote metaphase arrest and inactivation of Dbf2-2. After release from metaphase, overexpression of SIC1 was induced by addition of galactose to the culture medium to overcome the mitotic exit defects of dbf2-2 dbf20Δ cells. Samples were taken every 30 min to monitor Hof1 localization and actin repolarization. One representative cell (taken from time point 60 min) is shown in A–D . The levels of Hof1-GFP, Sic1, and Clb2 were monitored by immunoblotting using anti-GFP, anti-Sic1, and anti-Clb2 antibodies. Tub1 served as a loading control. The graphs indicate the percentage of cells ( n = 100–150) showing actin repolarization at the bud neck and Hof1-GFP at septins, at the AMR ring, or as a contracted ring.

    Techniques Used: Over Expression

    10) Product Images from "Misregulation of Scm3p/HJURP Causes Chromosome Instability in Saccharomyces cerevisiae and Human Cells"

    Article Title: Misregulation of Scm3p/HJURP Causes Chromosome Instability in Saccharomyces cerevisiae and Human Cells

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002303

    Overexpression of SCM3 causes reduced viability in a subset of kinetochore mutants and premature separation of sister chromatids. (A) GALSCM3 causes reduced viability in kinetochore mutants. A wild-type strain (Y5563) and kinetochore mutants ctf3Δ (YPH1712), mcm16Δ (YPH1714), mcm22Δ (YPH1716), ctf19Δ (YPH1713), mcm21Δ (YPH1715), okp1-5 (YPH1678), and ame1-4 (YPH1676) were transformed with GALSCM3HA (pMB1306) or vector (pRS426 GAL1 ). Equal number of cells from three independent transformants for each strain were plated on SC-URA with either glucose (2%) or galactose (2%). At least 2500 colonies were counted and % viability is expressed as the ratio of the number of colonies on galactose over the glucose media. (B) Overexpression of SCM3 leads to premature separation of sister chromatids in metaphase. Sister chromatid separation was monitored in nocodazole-arrested (metaphase) and alpha factor arrested (G1) cells by counting the number of GFP-LacI foci at the marked pericentromere of ChrIV in a wild-type (SBY818) and mcm21 Δ (SBY1897) strains overexpressing SCM3 (pMB1306) or a vector (pRS426 GAL1 ). At least 300 cells were analyzed for each strain. Pair-wise comparisons using Student's t -test were done to determine the statistical significance between samples. (C) Representative images of metaphase cells showing 1- and 2-GFP LacI foci (see, white arrows) are shown. (D) Overexpression of SCM3 causes lethality in ipl1-321 strain. Serial dilutions (5-fold) of ipl1-321 (SBY630) and wild type (SBY3) strains containing GALSCM3HA (pMB1306) or a vector (pRS426 GAL1 ) were plated on SC-URA with glucose (2%) or galactose (2%) plates and grown at 27°C and 33°C for 5 days.
    Figure Legend Snippet: Overexpression of SCM3 causes reduced viability in a subset of kinetochore mutants and premature separation of sister chromatids. (A) GALSCM3 causes reduced viability in kinetochore mutants. A wild-type strain (Y5563) and kinetochore mutants ctf3Δ (YPH1712), mcm16Δ (YPH1714), mcm22Δ (YPH1716), ctf19Δ (YPH1713), mcm21Δ (YPH1715), okp1-5 (YPH1678), and ame1-4 (YPH1676) were transformed with GALSCM3HA (pMB1306) or vector (pRS426 GAL1 ). Equal number of cells from three independent transformants for each strain were plated on SC-URA with either glucose (2%) or galactose (2%). At least 2500 colonies were counted and % viability is expressed as the ratio of the number of colonies on galactose over the glucose media. (B) Overexpression of SCM3 leads to premature separation of sister chromatids in metaphase. Sister chromatid separation was monitored in nocodazole-arrested (metaphase) and alpha factor arrested (G1) cells by counting the number of GFP-LacI foci at the marked pericentromere of ChrIV in a wild-type (SBY818) and mcm21 Δ (SBY1897) strains overexpressing SCM3 (pMB1306) or a vector (pRS426 GAL1 ). At least 300 cells were analyzed for each strain. Pair-wise comparisons using Student's t -test were done to determine the statistical significance between samples. (C) Representative images of metaphase cells showing 1- and 2-GFP LacI foci (see, white arrows) are shown. (D) Overexpression of SCM3 causes lethality in ipl1-321 strain. Serial dilutions (5-fold) of ipl1-321 (SBY630) and wild type (SBY3) strains containing GALSCM3HA (pMB1306) or a vector (pRS426 GAL1 ) were plated on SC-URA with glucose (2%) or galactose (2%) plates and grown at 27°C and 33°C for 5 days.

    Techniques Used: Over Expression, Transformation Assay, Plasmid Preparation

    11) Product Images from "Characterizing Cell Adhesion by Using Micropipette Aspiration"

    Article Title: Characterizing Cell Adhesion by Using Micropipette Aspiration

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2015.06.015

    Critical stress as a function of the aspiration rate and master equation. ( A ) Critical stress versus aspiration rate, from experimental data. Error bars represent the standard deviation. ( B ) Critical stress versus ln(( S pipette / S cell ) r P ) for the conditions where r P varies while S pipette / S cell is constant (data points shown in A ) (○), S pipette / S cell varies while r P is constant (●), cells are exposed to cytochalasin D (Δ), and cells are treated with nocodazole (★). All data collapse along the master curve, in good agreement with the prediction from Eq. 8. Error bars represent the standard error.
    Figure Legend Snippet: Critical stress as a function of the aspiration rate and master equation. ( A ) Critical stress versus aspiration rate, from experimental data. Error bars represent the standard deviation. ( B ) Critical stress versus ln(( S pipette / S cell ) r P ) for the conditions where r P varies while S pipette / S cell is constant (data points shown in A ) (○), S pipette / S cell varies while r P is constant (●), cells are exposed to cytochalasin D (Δ), and cells are treated with nocodazole (★). All data collapse along the master curve, in good agreement with the prediction from Eq. 8. Error bars represent the standard error.

    Techniques Used: Standard Deviation, Transferring

    12) Product Images from "Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration"

    Article Title: Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI64575

    Noscapine and taxol partially reverse nocodazole-induced MT disassembly in living cells. ( A ) Measurement of FDAP to determine MT dynamics in neuronal processes. PC12 cells were transiently transfected to express PAGFP-tagged tubulin, neuronally differentiated and focally irradiated with an UV laser in the middle of a process. FDAP, as an indicator for the ratio of soluble to polymerized tubulin, was determined in the activation spot, as indicated in the color-coded filled contour plots of 2D intensity function. ( B ) FDAP plots of cells treated with vehicle (0.01% DMSO), nocodazole alone, or nocodazole in combination with taxol or noscapine. Total fluorescence demonstrates photostability of the activated protein. Taxol and noscapine partially reversed the nocodazole-induced increase in FDAP, indicative of MT stabilization by these drugs. FDAP plots show the mean of n = 15–27 measurements per experimental condition.
    Figure Legend Snippet: Noscapine and taxol partially reverse nocodazole-induced MT disassembly in living cells. ( A ) Measurement of FDAP to determine MT dynamics in neuronal processes. PC12 cells were transiently transfected to express PAGFP-tagged tubulin, neuronally differentiated and focally irradiated with an UV laser in the middle of a process. FDAP, as an indicator for the ratio of soluble to polymerized tubulin, was determined in the activation spot, as indicated in the color-coded filled contour plots of 2D intensity function. ( B ) FDAP plots of cells treated with vehicle (0.01% DMSO), nocodazole alone, or nocodazole in combination with taxol or noscapine. Total fluorescence demonstrates photostability of the activated protein. Taxol and noscapine partially reversed the nocodazole-induced increase in FDAP, indicative of MT stabilization by these drugs. FDAP plots show the mean of n = 15–27 measurements per experimental condition.

    Techniques Used: Transfection, Irradiation, Activation Assay, Fluorescence

    13) Product Images from "Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration"

    Article Title: Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI64575

    Noscapine and taxol partially reverse nocodazole-induced MT disassembly in living cells. ( A ) Measurement of FDAP to determine MT dynamics in neuronal processes. PC12 cells were transiently transfected to express PAGFP-tagged tubulin, neuronally differentiated and focally irradiated with an UV laser in the middle of a process. FDAP, as an indicator for the ratio of soluble to polymerized tubulin, was determined in the activation spot, as indicated in the color-coded filled contour plots of 2D intensity function. ( B ) FDAP plots of cells treated with vehicle (0.01% DMSO), nocodazole alone, or nocodazole in combination with taxol or noscapine. Total fluorescence demonstrates photostability of the activated protein. Taxol and noscapine partially reversed the nocodazole-induced increase in FDAP, indicative of MT stabilization by these drugs. FDAP plots show the mean of n = 15–27 measurements per experimental condition.
    Figure Legend Snippet: Noscapine and taxol partially reverse nocodazole-induced MT disassembly in living cells. ( A ) Measurement of FDAP to determine MT dynamics in neuronal processes. PC12 cells were transiently transfected to express PAGFP-tagged tubulin, neuronally differentiated and focally irradiated with an UV laser in the middle of a process. FDAP, as an indicator for the ratio of soluble to polymerized tubulin, was determined in the activation spot, as indicated in the color-coded filled contour plots of 2D intensity function. ( B ) FDAP plots of cells treated with vehicle (0.01% DMSO), nocodazole alone, or nocodazole in combination with taxol or noscapine. Total fluorescence demonstrates photostability of the activated protein. Taxol and noscapine partially reversed the nocodazole-induced increase in FDAP, indicative of MT stabilization by these drugs. FDAP plots show the mean of n = 15–27 measurements per experimental condition.

    Techniques Used: Transfection, Irradiation, Activation Assay, Fluorescence

    14) Product Images from "Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry *"

    Article Title: Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.098558

    MLL5 is phosphorylated by Cdc2 at G 2 /M. A , mitotic HeLa cells were treated with various concentrations of roscovitine for 4 h in the presence of nocodazole. The phosphorylation of MLL5 was inhibited by roscovitine in a dose-dependent manner. B , asynchronous
    Figure Legend Snippet: MLL5 is phosphorylated by Cdc2 at G 2 /M. A , mitotic HeLa cells were treated with various concentrations of roscovitine for 4 h in the presence of nocodazole. The phosphorylation of MLL5 was inhibited by roscovitine in a dose-dependent manner. B , asynchronous

    Techniques Used:

    15) Product Images from "Assembling an intermediate filament network by dynamic cotranslation"

    Article Title: Assembling an intermediate filament network by dynamic cotranslation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200511033

    Insights into peripherin mRNA clustering. The relative roles of the nascent peripherin protein chain, the 3′UTR of peripherin mRNA, and MT in the formation of peripherin mRNA clusters were studied. (A) To determine the role of the nascent peripherin protein in mRNA clustering, a peripherin translation-null construct (ECFP-TAA-peripherin-R24-3′UTR) was created by inserting a stop codon (TAA) between the CFP and peripherin CDSs in the ECFP-peripherin-R24-3′UTR construct. To confirm the effectiveness of the inserted stop codon in preventing the translation of peripherin, Rat2 cells that are null for peripherin were transfected with either the ECFP-peripherin-R24-3′UTR construct as a control or the ECFP-TAA-peripherin-R24-3′UTR construct. After 24 h, whole cell lysates of these transfected Rat2 cells were separated by SDS-PAGE, transferred to nitrocellulose, and immuno-blotted with antibodies against CFP (GFP antibody) and peripherin. Vimentin, the endogenous Rat2 IF protein was used as a loading control. In cells expressing the control plasmid (CFP-Periph), both the peripherin and GFP antibodies recognized a product with a molecular weight of ∼81 kD, the predicted size of the CFP-peripherin fusion protein. In cells expressing the construct with the TAA insertion, there was no detectable peripherin by immunoblotting, and the GFP antibody recognized an ∼27-kD product, the predicted size of CFP. (B) A histogram comparing the distributions of peripherin mRNP clusters in control cells (blue bars, ECFP-peripherin-R24-3′UTR), ECFP-TAA-peripherin-R24-3′UTR–transfected cells (red bars), cells expressing the 3′UTR-null construct (yellow bars, ECFP-peripherin-R24), and control cells (ECFP-peripherin-R24-3′UTR) treated with nocodazole during NGF-induced differentiation (green bars). PC12 cells were transfected with the various constructs for 24 h, followed by NGF for 4 h. The cells were prepared for FISH and quantitatively analyzed as described in Fig. 4 . Error bars depict the SEM. (C) A histogram comparing the distributions of total peripherin mRNA in control cells (blue bars, ECFP-peripherin-R24-3′UTR); ECFP-TAA-peripherin-R24-3′UTR transfected cells (red bars); cells expressing the 3′UTR-null ECFP-peripherin-R24 construct (yellow bars); and control cells (ECFP-peripherin-R24-3′UTR) treated with nocodazole.
    Figure Legend Snippet: Insights into peripherin mRNA clustering. The relative roles of the nascent peripherin protein chain, the 3′UTR of peripherin mRNA, and MT in the formation of peripherin mRNA clusters were studied. (A) To determine the role of the nascent peripherin protein in mRNA clustering, a peripherin translation-null construct (ECFP-TAA-peripherin-R24-3′UTR) was created by inserting a stop codon (TAA) between the CFP and peripherin CDSs in the ECFP-peripherin-R24-3′UTR construct. To confirm the effectiveness of the inserted stop codon in preventing the translation of peripherin, Rat2 cells that are null for peripherin were transfected with either the ECFP-peripherin-R24-3′UTR construct as a control or the ECFP-TAA-peripherin-R24-3′UTR construct. After 24 h, whole cell lysates of these transfected Rat2 cells were separated by SDS-PAGE, transferred to nitrocellulose, and immuno-blotted with antibodies against CFP (GFP antibody) and peripherin. Vimentin, the endogenous Rat2 IF protein was used as a loading control. In cells expressing the control plasmid (CFP-Periph), both the peripherin and GFP antibodies recognized a product with a molecular weight of ∼81 kD, the predicted size of the CFP-peripherin fusion protein. In cells expressing the construct with the TAA insertion, there was no detectable peripherin by immunoblotting, and the GFP antibody recognized an ∼27-kD product, the predicted size of CFP. (B) A histogram comparing the distributions of peripherin mRNP clusters in control cells (blue bars, ECFP-peripherin-R24-3′UTR), ECFP-TAA-peripherin-R24-3′UTR–transfected cells (red bars), cells expressing the 3′UTR-null construct (yellow bars, ECFP-peripherin-R24), and control cells (ECFP-peripherin-R24-3′UTR) treated with nocodazole during NGF-induced differentiation (green bars). PC12 cells were transfected with the various constructs for 24 h, followed by NGF for 4 h. The cells were prepared for FISH and quantitatively analyzed as described in Fig. 4 . Error bars depict the SEM. (C) A histogram comparing the distributions of total peripherin mRNA in control cells (blue bars, ECFP-peripherin-R24-3′UTR); ECFP-TAA-peripherin-R24-3′UTR transfected cells (red bars); cells expressing the 3′UTR-null ECFP-peripherin-R24 construct (yellow bars); and control cells (ECFP-peripherin-R24-3′UTR) treated with nocodazole.

    Techniques Used: Construct, Transfection, SDS Page, Expressing, Plasmid Preparation, Molecular Weight, Fluorescence In Situ Hybridization

    FRAP analysis of a PC12 cell expressing GFP-peripherin. (A) A series of images of a single PC12 cell transfected with GFP-peripherin for 24 h, followed by NGF treatment for 4 h. This cell was photobleached in its entirety, and recovery of fluorescence was monitored by time-lapse imaging for a period of 10 min at 10-s intervals (Video 1). The cell, which is in the early stage of neurite extension, is depicted before photobleaching in i (phase and fluorescence) and ii (fluorescence). Particles were not seen immediately after photobleaching (iii), but were seen within 1 min after bleaching (iv). This rapid appearance of fluorescent particles after whole-cell photobleaching is completely inhibited by cycloheximide, as demonstrated by identical time-lapse observations over a 10-min period (Video 2 and not depicted). (B) The majority of peripherin particles move rapidly through the cytoplasm along microtubule tracks (Video 1; Helfand et al., 2004 ) making it extremely difficult to determine whether an individual fluorescent particle could recover its fluorescence. Therefore, PC12 cells were transfected with GFP-peripherin for 24 h, treated with NGF for 2 h, and exposed to nocodazole to disassemble microtubules and inhibit peripherin particle motility. In this experiment, one half of the cell (prebleach image shown in v) was left unbleached, as shown in the 10-s postbleach image (vi), to ensure that particles were immobilized. Time-lapse observations demonstrated that fluorescent particles appeared within 1 min (vii) and that after 10 min (viii) ∼30% of the particles ( n = 200) seen before photobleaching had recovered their fluorescence (Video 3). There was no significant increase in the number of particles that recovered after a longer recovery period of 30 min (not depicted). The 10-min postbleach image (viii) and the higher magnification image (ix) are displayed as an overlay between the prebleach (green) and the 10-min after bleach (red) images. Overlap between the green and the red appear yellow and represent particles that recovered their fluorescence. Not all the particles that recovered are yellow, but are red because of slight changes in particle position and shape over the 10-min observation period, leading to an incomplete overlap. Videos 1–3 are available at http://www.jcb.org/cgi/content/full/jcb.200511033/DC1 . Bars, 10 μm.
    Figure Legend Snippet: FRAP analysis of a PC12 cell expressing GFP-peripherin. (A) A series of images of a single PC12 cell transfected with GFP-peripherin for 24 h, followed by NGF treatment for 4 h. This cell was photobleached in its entirety, and recovery of fluorescence was monitored by time-lapse imaging for a period of 10 min at 10-s intervals (Video 1). The cell, which is in the early stage of neurite extension, is depicted before photobleaching in i (phase and fluorescence) and ii (fluorescence). Particles were not seen immediately after photobleaching (iii), but were seen within 1 min after bleaching (iv). This rapid appearance of fluorescent particles after whole-cell photobleaching is completely inhibited by cycloheximide, as demonstrated by identical time-lapse observations over a 10-min period (Video 2 and not depicted). (B) The majority of peripherin particles move rapidly through the cytoplasm along microtubule tracks (Video 1; Helfand et al., 2004 ) making it extremely difficult to determine whether an individual fluorescent particle could recover its fluorescence. Therefore, PC12 cells were transfected with GFP-peripherin for 24 h, treated with NGF for 2 h, and exposed to nocodazole to disassemble microtubules and inhibit peripherin particle motility. In this experiment, one half of the cell (prebleach image shown in v) was left unbleached, as shown in the 10-s postbleach image (vi), to ensure that particles were immobilized. Time-lapse observations demonstrated that fluorescent particles appeared within 1 min (vii) and that after 10 min (viii) ∼30% of the particles ( n = 200) seen before photobleaching had recovered their fluorescence (Video 3). There was no significant increase in the number of particles that recovered after a longer recovery period of 30 min (not depicted). The 10-min postbleach image (viii) and the higher magnification image (ix) are displayed as an overlay between the prebleach (green) and the 10-min after bleach (red) images. Overlap between the green and the red appear yellow and represent particles that recovered their fluorescence. Not all the particles that recovered are yellow, but are red because of slight changes in particle position and shape over the 10-min observation period, leading to an incomplete overlap. Videos 1–3 are available at http://www.jcb.org/cgi/content/full/jcb.200511033/DC1 . Bars, 10 μm.

    Techniques Used: Expressing, Transfection, Fluorescence, Imaging

    Observing peripherin particle formation in vivo. (A) A live cell cotransfected with both ECFP-peripherin-R24-3′UTR and YFP-MS2 for 24 h, followed by NGF treatment for 4 h, was treated with nocodazole for 30 min to depolymerize microtubules. This treatment inhibited the motility of both peripherin particles and mRNPs, thereby facilitating the observation of peripherin particle formation in association with mRNPs. Time-lapse imaging shows an mRNP (red) that appears to be engaged in protein synthesis (green; arrowhead) in a thin peripheral region of the cytoplasm (Video 8). The green signal appears first as a dim haze (i and ii) that becomes brighter and more distinct with time (iii), resulting in the formation of a typical IF particle by 180 s (iv). (B) The association of ribosomes with peripherin mRNPs. Cells expressing both ECFP-peripherin-R24-3′UTR and YFP-MS2 were processed for indirect immunofluorescence using an antibody against the S6 subunit of ribosomes. Approximately 70% of peripherin mRNPs (vi, light blue) associated with peripherin protein particles (v; green) were also associated with ribosomes (vii, red) as shown in the triple overlay in viii. (C) Distribution of peripherin particles. 29% of peripherin particles are associated with peripherin mRNA. Of this fraction, ∼70% are also associated with ribosomes. The remaining 30% may represent complexes in which the translation process has ceased and the ribosomes have dissociated. Video 8 is available at http://www.jcb.org/cgi/content/full/jcb.200511033/DC1 . Bars, 5 μm.
    Figure Legend Snippet: Observing peripherin particle formation in vivo. (A) A live cell cotransfected with both ECFP-peripherin-R24-3′UTR and YFP-MS2 for 24 h, followed by NGF treatment for 4 h, was treated with nocodazole for 30 min to depolymerize microtubules. This treatment inhibited the motility of both peripherin particles and mRNPs, thereby facilitating the observation of peripherin particle formation in association with mRNPs. Time-lapse imaging shows an mRNP (red) that appears to be engaged in protein synthesis (green; arrowhead) in a thin peripheral region of the cytoplasm (Video 8). The green signal appears first as a dim haze (i and ii) that becomes brighter and more distinct with time (iii), resulting in the formation of a typical IF particle by 180 s (iv). (B) The association of ribosomes with peripherin mRNPs. Cells expressing both ECFP-peripherin-R24-3′UTR and YFP-MS2 were processed for indirect immunofluorescence using an antibody against the S6 subunit of ribosomes. Approximately 70% of peripherin mRNPs (vi, light blue) associated with peripherin protein particles (v; green) were also associated with ribosomes (vii, red) as shown in the triple overlay in viii. (C) Distribution of peripherin particles. 29% of peripherin particles are associated with peripherin mRNA. Of this fraction, ∼70% are also associated with ribosomes. The remaining 30% may represent complexes in which the translation process has ceased and the ribosomes have dissociated. Video 8 is available at http://www.jcb.org/cgi/content/full/jcb.200511033/DC1 . Bars, 5 μm.

    Techniques Used: In Vivo, Imaging, Expressing, Immunofluorescence

    16) Product Images from "Cdk1 phosphorylates the Rac activator Tiam1 to activate centrosomal Pak and promote mitotic spindle formation"

    Article Title: Cdk1 phosphorylates the Rac activator Tiam1 to activate centrosomal Pak and promote mitotic spindle formation

    Journal: Nature Communications

    doi: 10.1038/ncomms8437

    Cdk1-dependent phosphorylation of Tiam1 in mitosis. ( a ) MDCK II cells were synchronized in G1, S or G2 phases as described in Methods or in mitosis (M) by nocodazole treatment. Lysates were prepared and protein levels analysed by immunoblotting with the indicated antibodies. ( b ) Lysates were prepared from untreated or nocodazole treated MDCK II cells, treated with lambda phosphatase ( λ -PPase) where indicated and analysed by immunoblotting. ( c ) MDCK II cells were left asynchronous or arrested in mitosis (STLC) then treated with RO-3306 (10 μM) where indicated for 3 h before lysis and immunoblotting. In a and c β-actin was used as a loading control. ( d ) Tiam1-HA was immunoprecipitated from HEK293T cells then subjected to in vitro kinase assay with ATP and GST-tagged Cdk1-cyclin B1 complex as indicated. Following SDS–PAGE, phosphorylation was measured by immunoblotting with anti-P*-Thr-Pro antibody (P*S/T-P). ( e ) Purified Tiam1-His was used for in vitro kinase assay with GST-tagged Cdk1-cyclin B1 and analysed as in d . ( f ) Tiam1-HA (either WT or the S1466A mutant) was immunoprecipitated from HEK293T cells arrested in mitosis (STLC) and analysed by immunoblotting with P*S/T-P antibody. Quantitation shows mean P*S/T-P normalized to HA signal+s.e.m. (with WT set as 1) ( n =4, unpaired two-sided t -test: * P
    Figure Legend Snippet: Cdk1-dependent phosphorylation of Tiam1 in mitosis. ( a ) MDCK II cells were synchronized in G1, S or G2 phases as described in Methods or in mitosis (M) by nocodazole treatment. Lysates were prepared and protein levels analysed by immunoblotting with the indicated antibodies. ( b ) Lysates were prepared from untreated or nocodazole treated MDCK II cells, treated with lambda phosphatase ( λ -PPase) where indicated and analysed by immunoblotting. ( c ) MDCK II cells were left asynchronous or arrested in mitosis (STLC) then treated with RO-3306 (10 μM) where indicated for 3 h before lysis and immunoblotting. In a and c β-actin was used as a loading control. ( d ) Tiam1-HA was immunoprecipitated from HEK293T cells then subjected to in vitro kinase assay with ATP and GST-tagged Cdk1-cyclin B1 complex as indicated. Following SDS–PAGE, phosphorylation was measured by immunoblotting with anti-P*-Thr-Pro antibody (P*S/T-P). ( e ) Purified Tiam1-His was used for in vitro kinase assay with GST-tagged Cdk1-cyclin B1 and analysed as in d . ( f ) Tiam1-HA (either WT or the S1466A mutant) was immunoprecipitated from HEK293T cells arrested in mitosis (STLC) and analysed by immunoblotting with P*S/T-P antibody. Quantitation shows mean P*S/T-P normalized to HA signal+s.e.m. (with WT set as 1) ( n =4, unpaired two-sided t -test: * P

    Techniques Used: Lysis, Immunoprecipitation, In Vitro, Kinase Assay, SDS Page, Purification, Mutagenesis, Quantitation Assay

    17) Product Images from "The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis"

    Article Title: The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200807023

    CLIP-170 and mDia1 interaction does not depend on actin or microtubules and is regulated during phagocytosis. (A) Cells were treated with 10 μM taxol, 0.1 μM latrunculin A, or 0.5 μM nocodazole during 45 min and then lysed. Lysates were analyzed as in Fig. 5 A . Data are representative of four independent experiments. (B and C) The mDia1–CLIP-170 complex is regulated during CR3- (B) but not FcR-mediated (C) phagocytosis. RAW264.7 macrophages were incubated with nonopsonized (control), C3bi-, or IgG-SRBCs for various times at 37°C. Lysates were incubated with monoclonal anti–CLIP-170 antibodies or an irrelevant antibody (IrAb). Western blots were performed using anti-mDia1 antibodies, and then with anti–CLIP-170/CLIP-115 antibodies (#2221 serum). The intensities of the bands were analyzed as in Fig. 5 F except that the ratio was calculated for each time point as compared with the control. Data are representative of three to four independent experiments. *, P
    Figure Legend Snippet: CLIP-170 and mDia1 interaction does not depend on actin or microtubules and is regulated during phagocytosis. (A) Cells were treated with 10 μM taxol, 0.1 μM latrunculin A, or 0.5 μM nocodazole during 45 min and then lysed. Lysates were analyzed as in Fig. 5 A . Data are representative of four independent experiments. (B and C) The mDia1–CLIP-170 complex is regulated during CR3- (B) but not FcR-mediated (C) phagocytosis. RAW264.7 macrophages were incubated with nonopsonized (control), C3bi-, or IgG-SRBCs for various times at 37°C. Lysates were incubated with monoclonal anti–CLIP-170 antibodies or an irrelevant antibody (IrAb). Western blots were performed using anti-mDia1 antibodies, and then with anti–CLIP-170/CLIP-115 antibodies (#2221 serum). The intensities of the bands were analyzed as in Fig. 5 F except that the ratio was calculated for each time point as compared with the control. Data are representative of three to four independent experiments. *, P

    Techniques Used: Cross-linking Immunoprecipitation, Incubation, Western Blot

    18) Product Images from "Zwint-1 is required for spindle assembly checkpoint function and kinetochore-microtubule attachment during oocyte meiosis"

    Article Title: Zwint-1 is required for spindle assembly checkpoint function and kinetochore-microtubule attachment during oocyte meiosis

    Journal: Scientific Reports

    doi: 10.1038/srep15431

    Mad2-mediated SAC inactivation in Zwint-1-knockdown oocytes. ( a ) Oocytes injected with the indicated dsRNA were cultured with 400 nM nocodazole from 4 h after GVBD and the PBE rate was scored at 13 h after GVBD. Data are mean ± SEM from three independent experiments (* p
    Figure Legend Snippet: Mad2-mediated SAC inactivation in Zwint-1-knockdown oocytes. ( a ) Oocytes injected with the indicated dsRNA were cultured with 400 nM nocodazole from 4 h after GVBD and the PBE rate was scored at 13 h after GVBD. Data are mean ± SEM from three independent experiments (* p

    Techniques Used: Injection, Cell Culture

    19) Product Images from "Method to Synchronize Cell Cycle of Human Pluripotent Stem Cells without Affecting Their Fundamental Characteristics"

    Article Title: Method to Synchronize Cell Cycle of Human Pluripotent Stem Cells without Affecting Their Fundamental Characteristics

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2018.11.020

    Single-Cell RNA-Seq Confirms that Nocodazole Treatment Does Not Affect the Ability of Pluripotent Cells to Differentiate into Definitive Endoderm (A) Schematic overview of experimental setup for performing single-cell RNA-seq analysis on pluripotent and endoderm cells following nocodazole treatment. (B) Plots showing two projections of a 3D t-SNE embedding. Dots represent individual cells. Cells were labeled based on their differentiation and synchronization status. Normalized log-expression values were used (DMSO = Green, nocodazole [Noc] = Purple, pluripotent [Pluri] = Circle, Endoderm = Triangle). (C) t-SNE plot showing the expression pattern of pluripotency ( POUF51 , NANOG , SOX2 ) and endoderm ( SOX17 , GATA6 , CER1 ) genes in each cluster. Dots represent individual cells. (D) t-SNE plot showing the assignment of clusters identified by applying an SNN modularity optimization algorithm (see Experimental Procedures ) in DMSO- and Noc-treated cells. Normalized log-expression values were used. Dots represent individual cells (DMSO = Circle, Noc = Triangle). (E) Heatmap showing the list of 50 differentially expressed genes obtained when merging the 10 genes with highest average log fold change in each cluster. Clusters 0, 3, and 4 represent undifferentiated cells and clusters 1 and 2 endoderm cells. (F) Scatterplot showing the log-average expression in cluster 1 versus cluster 2. Genes differentially expressed among cluster 1 and 2 are highlighted in light gray and red, with red representing genes with a log2FC ≥ 1. Genes that are not differentially expressed among these two groups are highlighted in green. (G) GO analyses of clusters 1 and 2 for the genes found in either cluster 1 (cluster 1 unique) or cluster 2 (cluster 2 unique), as well as for those genes that are not differentially expressed between these two groups (clusters 1 and 2). See also Figure S3 .
    Figure Legend Snippet: Single-Cell RNA-Seq Confirms that Nocodazole Treatment Does Not Affect the Ability of Pluripotent Cells to Differentiate into Definitive Endoderm (A) Schematic overview of experimental setup for performing single-cell RNA-seq analysis on pluripotent and endoderm cells following nocodazole treatment. (B) Plots showing two projections of a 3D t-SNE embedding. Dots represent individual cells. Cells were labeled based on their differentiation and synchronization status. Normalized log-expression values were used (DMSO = Green, nocodazole [Noc] = Purple, pluripotent [Pluri] = Circle, Endoderm = Triangle). (C) t-SNE plot showing the expression pattern of pluripotency ( POUF51 , NANOG , SOX2 ) and endoderm ( SOX17 , GATA6 , CER1 ) genes in each cluster. Dots represent individual cells. (D) t-SNE plot showing the assignment of clusters identified by applying an SNN modularity optimization algorithm (see Experimental Procedures ) in DMSO- and Noc-treated cells. Normalized log-expression values were used. Dots represent individual cells (DMSO = Circle, Noc = Triangle). (E) Heatmap showing the list of 50 differentially expressed genes obtained when merging the 10 genes with highest average log fold change in each cluster. Clusters 0, 3, and 4 represent undifferentiated cells and clusters 1 and 2 endoderm cells. (F) Scatterplot showing the log-average expression in cluster 1 versus cluster 2. Genes differentially expressed among cluster 1 and 2 are highlighted in light gray and red, with red representing genes with a log2FC ≥ 1. Genes that are not differentially expressed among these two groups are highlighted in green. (G) GO analyses of clusters 1 and 2 for the genes found in either cluster 1 (cluster 1 unique) or cluster 2 (cluster 2 unique), as well as for those genes that are not differentially expressed between these two groups (clusters 1 and 2). See also Figure S3 .

    Techniques Used: RNA Sequencing Assay, Labeling, Expressing

    Nocodazole Treatment Does Not Affect the Capacity of hESCs to Differentiate into the Three Germ Layers (A) Schematic representation of the in vitro differentiation protocol to generate the three mesoderm subtypes lateral plate mesoderm (LPM), cardiac mesoderm (CM), and presomitic mesoderm (PSM) as well as endoderm and ectoderm. Treatment for the induction of the three mesoderm subtypes LPM, CM, and PSM is for 36 hr. Treatment for generation of endoderm is for 3 days and for ectoderm 6 days. (B–F) Immunostaining analysis for BRACHYURY expression during LPM induction (B), EOMES expression during CM induction (C), BRACHYURY expression during PSM induction (D), SOX17 expression in definitive endoderm (E), and SOX1 expression in ectoderm cells (F). Scale bars, 100 μm.
    Figure Legend Snippet: Nocodazole Treatment Does Not Affect the Capacity of hESCs to Differentiate into the Three Germ Layers (A) Schematic representation of the in vitro differentiation protocol to generate the three mesoderm subtypes lateral plate mesoderm (LPM), cardiac mesoderm (CM), and presomitic mesoderm (PSM) as well as endoderm and ectoderm. Treatment for the induction of the three mesoderm subtypes LPM, CM, and PSM is for 36 hr. Treatment for generation of endoderm is for 3 days and for ectoderm 6 days. (B–F) Immunostaining analysis for BRACHYURY expression during LPM induction (B), EOMES expression during CM induction (C), BRACHYURY expression during PSM induction (D), SOX17 expression in definitive endoderm (E), and SOX1 expression in ectoderm cells (F). Scale bars, 100 μm.

    Techniques Used: In Vitro, Immunostaining, Expressing

    Cell Cycle Synchronization Is Partially Maintained after Release from Nocodazole Inhibition (A) Cell cycle profile of H9 hESCs following release from nocodazole inhibition. Samples were analyzed through a time course of 24 hr. (B) Bar graph summarizing the flow cytometry cell cycle profile analysis of H9 hESCs. Error bars represent ±SEM of five independent experiments. (C) Western blot for cyclin D1, cyclin D2, and cyclin D3 proteins in H9 hESCs through a time course of 24 hr following nocodazole release.
    Figure Legend Snippet: Cell Cycle Synchronization Is Partially Maintained after Release from Nocodazole Inhibition (A) Cell cycle profile of H9 hESCs following release from nocodazole inhibition. Samples were analyzed through a time course of 24 hr. (B) Bar graph summarizing the flow cytometry cell cycle profile analysis of H9 hESCs. Error bars represent ±SEM of five independent experiments. (C) Western blot for cyclin D1, cyclin D2, and cyclin D3 proteins in H9 hESCs through a time course of 24 hr following nocodazole release.

    Techniques Used: Inhibition, Flow Cytometry, Cytometry, Western Blot

    Human iPSCs Can Be Synchronized with Nocodazole while Maintaining Their Capacity of Differentiation (A–C) Cell cycle profile of hiPSCs lines FSPS13B (A), CF04 (B), and CF05 (C) following treatment and release from nocodazole. (D–F) Immunostaining analysis for the expression of early mesoderm markers HAND1 (during LPM induction, D), EOMES (during CM induction, E), and BRACHYURY (during PSM induction, F) in DMSO- and nocodazole-treated cells after 36 hr of differentiation. Scale bar, 100 μm. See also Figure S4 .
    Figure Legend Snippet: Human iPSCs Can Be Synchronized with Nocodazole while Maintaining Their Capacity of Differentiation (A–C) Cell cycle profile of hiPSCs lines FSPS13B (A), CF04 (B), and CF05 (C) following treatment and release from nocodazole. (D–F) Immunostaining analysis for the expression of early mesoderm markers HAND1 (during LPM induction, D), EOMES (during CM induction, E), and BRACHYURY (during PSM induction, F) in DMSO- and nocodazole-treated cells after 36 hr of differentiation. Scale bar, 100 μm. See also Figure S4 .

    Techniques Used: Immunostaining, Expressing

    Nocodazole Treatment Does Not Affect Pluripotency of hESCs (A) Brightfield images of H9 hESCs showing cell morphology after nocodazole release. Scale bars, 400 μm. (B) qRT-PCR analysis for pluripotency and differentiation markers in H9 hESCs through a time course of 24 hr after nocodazole release. Error bars represent ±SEM of two independent experiments. (C) qRT-PCR analysis for pluripotency and differentiation markers in H9 hESCs at passage 2, passage 3, and passage 16 after nocodazole treatment. Error bars represent ±SEM of triplicates in an independent experiment. (D) Representative flow cytometry analysis for OCT4 expression in H9 hESCs through a time course of 24 hr after nocodazole release. (E) Immunostaining analysis for the expression of pluripotency markers OCT4, NANOG, and SOX2 and differentiation markers BRACHYURY, EOMES, and SOX1 in DMSO- and nocodazole-treated H9 hESCs, two passages after nocodazole release. Scale bar, 200 μm. See also Figure S2 .
    Figure Legend Snippet: Nocodazole Treatment Does Not Affect Pluripotency of hESCs (A) Brightfield images of H9 hESCs showing cell morphology after nocodazole release. Scale bars, 400 μm. (B) qRT-PCR analysis for pluripotency and differentiation markers in H9 hESCs through a time course of 24 hr after nocodazole release. Error bars represent ±SEM of two independent experiments. (C) qRT-PCR analysis for pluripotency and differentiation markers in H9 hESCs at passage 2, passage 3, and passage 16 after nocodazole treatment. Error bars represent ±SEM of triplicates in an independent experiment. (D) Representative flow cytometry analysis for OCT4 expression in H9 hESCs through a time course of 24 hr after nocodazole release. (E) Immunostaining analysis for the expression of pluripotency markers OCT4, NANOG, and SOX2 and differentiation markers BRACHYURY, EOMES, and SOX1 in DMSO- and nocodazole-treated H9 hESCs, two passages after nocodazole release. Scale bar, 200 μm. See also Figure S2 .

    Techniques Used: Quantitative RT-PCR, Flow Cytometry, Cytometry, Expressing, Immunostaining

    Nocodazole Is the Most Efficient Small-Molecule Inhibitor to Synchronize the Cell Cycle in hPSCs (A) Schematic showing the cell cycle phase inhibited by small molecules. (B) Schematic overview of the experimental setup to determine the efficiency of each small molecule for synchronizing the cell cycle of hESCs. (C) Brightfield images of colony morphology of H9 hESCs after 16 hr of treatment with the different small molecule cell cycle inhibitors. Scale bars, 400 μm. (D–G) Cell cycle profile of H9 hESCs following treatment and removal of cell cycle inhibitors thymidine (D), aphidicolin (E), hydroxyurea (F), and nocodazole (G) through a time course of 24 hr. See also Figure S1 .
    Figure Legend Snippet: Nocodazole Is the Most Efficient Small-Molecule Inhibitor to Synchronize the Cell Cycle in hPSCs (A) Schematic showing the cell cycle phase inhibited by small molecules. (B) Schematic overview of the experimental setup to determine the efficiency of each small molecule for synchronizing the cell cycle of hESCs. (C) Brightfield images of colony morphology of H9 hESCs after 16 hr of treatment with the different small molecule cell cycle inhibitors. Scale bars, 400 μm. (D–G) Cell cycle profile of H9 hESCs following treatment and removal of cell cycle inhibitors thymidine (D), aphidicolin (E), hydroxyurea (F), and nocodazole (G) through a time course of 24 hr. See also Figure S1 .

    Techniques Used:

    hESCs Can Generate Functional Cell Types following Nocodazole Treatment (A) qRT-PCR analysis for SMC markers in DMSO and nocodazole-treated cells. Error bars represent ±SEM of three independent experiments. Ordinary one-way ANOVA test followed by Sidak's test for comparison of DMSO versus nocodazole-treated cells was performed. (ns, not significant). (B) Contractility of SMCs was induced by carbachol. Panels show cells contracting within 10 min of carbachol treatment. Graph shows % contraction of 20 cells in DMSO control and nocodazole-treated cells. Error bars represent ±SEM. (C) qRT-PCR analysis for cardiomyocyte markers in DMSO and nocodazole-treated cells. Error bars represent ±SEM of three independent experiments. Ordinary one-way ANOVA test followed by Sidak's test for comparison of DMSO versus nocodazole-treated cells was performed (ns, not significant). (D) Graph showing beating rate of cardiomyocytes generated form DMSO and nocodazole-treated cells. Error bars represent ±SEM (n = 4). (E) qRT-PCR analysis for chondrocyte markers in DMSO- and nocodazole-treated cells. Error bars represent ±SEM of two independent experiments. (F) Alcian blue staining of chondrocytes shows Alcian blue absorption and release of DMSO control and nocodazole-treated cells. Error bars represent ±SEM of triplicates in an independent experiment. (G) qRT-PCR analysis for hepatocyte markers in DMSO and nocodazole-treated cells. Error bars represent ±SEM of triplicates in an independent experiment. (H) Hepatocytes generated from DMSO and nocodazole-treated cells display cytochrome P450 3A4 activity, as assessed by the enzymatic release of free luciferin by cytochrome P450 from an inactive luciferin precursor. Error bars represent ±SEM of triplicates in an independent experiment.
    Figure Legend Snippet: hESCs Can Generate Functional Cell Types following Nocodazole Treatment (A) qRT-PCR analysis for SMC markers in DMSO and nocodazole-treated cells. Error bars represent ±SEM of three independent experiments. Ordinary one-way ANOVA test followed by Sidak's test for comparison of DMSO versus nocodazole-treated cells was performed. (ns, not significant). (B) Contractility of SMCs was induced by carbachol. Panels show cells contracting within 10 min of carbachol treatment. Graph shows % contraction of 20 cells in DMSO control and nocodazole-treated cells. Error bars represent ±SEM. (C) qRT-PCR analysis for cardiomyocyte markers in DMSO and nocodazole-treated cells. Error bars represent ±SEM of three independent experiments. Ordinary one-way ANOVA test followed by Sidak's test for comparison of DMSO versus nocodazole-treated cells was performed (ns, not significant). (D) Graph showing beating rate of cardiomyocytes generated form DMSO and nocodazole-treated cells. Error bars represent ±SEM (n = 4). (E) qRT-PCR analysis for chondrocyte markers in DMSO- and nocodazole-treated cells. Error bars represent ±SEM of two independent experiments. (F) Alcian blue staining of chondrocytes shows Alcian blue absorption and release of DMSO control and nocodazole-treated cells. Error bars represent ±SEM of triplicates in an independent experiment. (G) qRT-PCR analysis for hepatocyte markers in DMSO and nocodazole-treated cells. Error bars represent ±SEM of triplicates in an independent experiment. (H) Hepatocytes generated from DMSO and nocodazole-treated cells display cytochrome P450 3A4 activity, as assessed by the enzymatic release of free luciferin by cytochrome P450 from an inactive luciferin precursor. Error bars represent ±SEM of triplicates in an independent experiment.

    Techniques Used: Functional Assay, Quantitative RT-PCR, Generated, Staining, Activity Assay

    20) Product Images from "Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1"

    Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06854-2

    Expression of ASXL2 K370R reduces mammalian cell proliferation. a Enforced expression of ASXL2 K370R decreases cellular proliferation. U-2 OS cells were transduced with different amounts of lentiviral suspensions produced using ASXL2 or ASXL2 K370R constructs. Cells were selected by puromycin and harvested for immunoblotting (top panel). Equal numbers of puromycin-selected cells were plated for colony formation assay (CFA) (bottom panel). n = 2 biological replicates. b The cells infected in a , were treated with nocodazole for FACS analysis at the indicated times. Note that (+2×) refers to transduction of cells with twice the amount of virus we normally use for Myc-ASXL2, and (−2×) refers to transduction of the cells with two times less the amount of viruses we normally use for ASXL2 370R. This adjustment was conducted to correct for the expression levels usually higher for ASXL2 K370R. n = 2 biological replicates. c , d Normal diploid fibroblast IMR90 cells were transduced with viral expression constructs for ASXL2 or ASXL2 K370R. Cells were selected by puromycin and equal numbers were plated for phase contrast pictures ( c ) or cell counts ( d ). Scale bar: 50 µm for panel c . n = 2 biological replicates. (Exp.1 and Exp.2). e siRNA depletion of ASXL2 decreases cellular proliferation. U-2 OS cells were transfected with NT siRNA control or siRNA for ASXL2 . Equal numbers of puromycin-selected cells were plated for CFA (left panel). Cells were treated with nocodazole for FACS analysis (right panel). f siRNA depletion of UBE2E3 decreases cellular proliferation. U-2 OS cells were transfected with individual siRNA constructs as indicated. Cells were plated for viability measurement using MTT assay. n = 3 biological replicates. Error bars represent s.d. (mean ± SD). g , h Inactivation of UBE2E3 locus decreases cellular proliferation. Schematic representation for gRNAs targeting the UBE2E3 locus ( g top panel). U-2 OS cells were transduced with different lentiviral CRISPR/Cas9 constructs, selected by puromycin and harvested for immunoblotting ( g bottom panel). n = 3 biological replicates. Equal numbers of puromycin-selected cells were plated for CFA ( h ). n = 2 biological replicates. i The cells selected as in h were treated with nocodazole for FACS analysis at the indicated time . n = 2 biological replicates. j Positive correlation of BAP1, ASXL2, and UBE2E3 protein expression levels in human mesothelioma. Mesothelioma biopsies were immunostained for ASXL2, UBE2E3, or BAP1 (see Supplementary Fig. 11a and Supplementary Table 3 ). Pictures were taken at 100× magnification. Scale bar: 100 μm. Tubulin was used as a loading control for panels a and g
    Figure Legend Snippet: Expression of ASXL2 K370R reduces mammalian cell proliferation. a Enforced expression of ASXL2 K370R decreases cellular proliferation. U-2 OS cells were transduced with different amounts of lentiviral suspensions produced using ASXL2 or ASXL2 K370R constructs. Cells were selected by puromycin and harvested for immunoblotting (top panel). Equal numbers of puromycin-selected cells were plated for colony formation assay (CFA) (bottom panel). n = 2 biological replicates. b The cells infected in a , were treated with nocodazole for FACS analysis at the indicated times. Note that (+2×) refers to transduction of cells with twice the amount of virus we normally use for Myc-ASXL2, and (−2×) refers to transduction of the cells with two times less the amount of viruses we normally use for ASXL2 370R. This adjustment was conducted to correct for the expression levels usually higher for ASXL2 K370R. n = 2 biological replicates. c , d Normal diploid fibroblast IMR90 cells were transduced with viral expression constructs for ASXL2 or ASXL2 K370R. Cells were selected by puromycin and equal numbers were plated for phase contrast pictures ( c ) or cell counts ( d ). Scale bar: 50 µm for panel c . n = 2 biological replicates. (Exp.1 and Exp.2). e siRNA depletion of ASXL2 decreases cellular proliferation. U-2 OS cells were transfected with NT siRNA control or siRNA for ASXL2 . Equal numbers of puromycin-selected cells were plated for CFA (left panel). Cells were treated with nocodazole for FACS analysis (right panel). f siRNA depletion of UBE2E3 decreases cellular proliferation. U-2 OS cells were transfected with individual siRNA constructs as indicated. Cells were plated for viability measurement using MTT assay. n = 3 biological replicates. Error bars represent s.d. (mean ± SD). g , h Inactivation of UBE2E3 locus decreases cellular proliferation. Schematic representation for gRNAs targeting the UBE2E3 locus ( g top panel). U-2 OS cells were transduced with different lentiviral CRISPR/Cas9 constructs, selected by puromycin and harvested for immunoblotting ( g bottom panel). n = 3 biological replicates. Equal numbers of puromycin-selected cells were plated for CFA ( h ). n = 2 biological replicates. i The cells selected as in h were treated with nocodazole for FACS analysis at the indicated time . n = 2 biological replicates. j Positive correlation of BAP1, ASXL2, and UBE2E3 protein expression levels in human mesothelioma. Mesothelioma biopsies were immunostained for ASXL2, UBE2E3, or BAP1 (see Supplementary Fig. 11a and Supplementary Table 3 ). Pictures were taken at 100× magnification. Scale bar: 100 μm. Tubulin was used as a loading control for panels a and g

    Techniques Used: Expressing, Transduction, Produced, Construct, Colony Assay, Infection, FACS, Transfection, MTT Assay, CRISPR

    21) Product Images from "Phosphorylation and Stabilization of HURP by Aurora-A: Implication of HURP as a Transforming Target of Aurora-A"

    Article Title: Phosphorylation and Stabilization of HURP by Aurora-A: Implication of HURP as a Transforming Target of Aurora-A

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.14.5789-5800.2005

    Aurora-A protects HURP from protein degradation. 293T cells were transiently transfected with HA-HURP-WT or HA-HURP-4P. Twenty-four hours after transfection, the 293T cells were synchronized in mitosis by nocodazole (A) or monastrol (C) treatment for 16 h. Cells were released into cell cycle progression by removing these mitosis-synchronizing agents and subsequently incubated with fresh medium containing cycloheximide, which blocks de novo protein synthesis. At the indicated time points, cells were harvested and analyzed by Western blotting (WB) with anti-HA antibody. (B) 293T cells were transiently transfected with the pCMV2 vector (vehicle), FLAG-Aurora-A-WT, or FLAG-Aurora-A-KD; synchronized in mitosis by nocodazole treatment; and released from nocodazole blockage as described for panel A. At the indicated time points, the cells were harvested and analyzed by Western blotting with antibodies against HURP, FLAG, cyclin B1, and actin. Cyclin B1 is well known to be degraded at the M/G 1 transition, and the rapid decrease in cyclin B1 serves as a positive control showing that protein degradation under these conditions is working normally. Actin was used as a loading control. The results shown are representative of three independent experiments.
    Figure Legend Snippet: Aurora-A protects HURP from protein degradation. 293T cells were transiently transfected with HA-HURP-WT or HA-HURP-4P. Twenty-four hours after transfection, the 293T cells were synchronized in mitosis by nocodazole (A) or monastrol (C) treatment for 16 h. Cells were released into cell cycle progression by removing these mitosis-synchronizing agents and subsequently incubated with fresh medium containing cycloheximide, which blocks de novo protein synthesis. At the indicated time points, cells were harvested and analyzed by Western blotting (WB) with anti-HA antibody. (B) 293T cells were transiently transfected with the pCMV2 vector (vehicle), FLAG-Aurora-A-WT, or FLAG-Aurora-A-KD; synchronized in mitosis by nocodazole treatment; and released from nocodazole blockage as described for panel A. At the indicated time points, the cells were harvested and analyzed by Western blotting with antibodies against HURP, FLAG, cyclin B1, and actin. Cyclin B1 is well known to be degraded at the M/G 1 transition, and the rapid decrease in cyclin B1 serves as a positive control showing that protein degradation under these conditions is working normally. Actin was used as a loading control. The results shown are representative of three independent experiments.

    Techniques Used: Transfection, Incubation, Western Blot, Plasmid Preparation, Positive Control

    HURP is a potential substrate of Aurora-A. (A) Recombinant Aurora-A was incubated with recombinant His-tagged HURP in kinase reaction buffer in the presence of [γ- 32 P]ATP for different time intervals. The samples were separated by SDS-PAGE and transferred to a PVDF membrane, and this was followed by autoradiography (top) or membrane staining with Coomassie blue (bottom). An asterisk indicates multiple phosphorylation forms of HURP. (B) Recombinant Aurora-A, Aurora-B, and Aurora-C were incubated with recombinant His-tagged HURP in the presence of [γ- 32 P]ATP. Analysis of the autoradiography data demonstrates that Aurora-A efficiently phosphorylated the recombinant HURP; however, no such phosphorylation was observed when Aurora-B or Aurora-C was present. Histone H1, histone H2A, myelin basic protein (MBP), and p16 were also incubated with the Aurora kinases to serve as substrate controls and ensure that the input of Aurora-A kinase activity was similar to that of Aurora-B and that the Aurora-C used was active. (C) 293T cells were transfected with pSUPER vector (vehicle) or pSUPER-Aurora-A for 48 h and then synchronized in mitosis using nocodazole treatment for 16 h. Equal amounts of extracts were prepared and analyzed by Western blotting (WB) with anti-Aurora-A, anti-HURP, anti-cyclin B1, or anti-actin antibodies. Actin was used as a loading control. The similar cyclin B1 expression level indicates a comparable synchronization effect for pSUPER-Aurora-A transfectants during mitosis. The asterisk indicates the phosphorylation form of HURP.
    Figure Legend Snippet: HURP is a potential substrate of Aurora-A. (A) Recombinant Aurora-A was incubated with recombinant His-tagged HURP in kinase reaction buffer in the presence of [γ- 32 P]ATP for different time intervals. The samples were separated by SDS-PAGE and transferred to a PVDF membrane, and this was followed by autoradiography (top) or membrane staining with Coomassie blue (bottom). An asterisk indicates multiple phosphorylation forms of HURP. (B) Recombinant Aurora-A, Aurora-B, and Aurora-C were incubated with recombinant His-tagged HURP in the presence of [γ- 32 P]ATP. Analysis of the autoradiography data demonstrates that Aurora-A efficiently phosphorylated the recombinant HURP; however, no such phosphorylation was observed when Aurora-B or Aurora-C was present. Histone H1, histone H2A, myelin basic protein (MBP), and p16 were also incubated with the Aurora kinases to serve as substrate controls and ensure that the input of Aurora-A kinase activity was similar to that of Aurora-B and that the Aurora-C used was active. (C) 293T cells were transfected with pSUPER vector (vehicle) or pSUPER-Aurora-A for 48 h and then synchronized in mitosis using nocodazole treatment for 16 h. Equal amounts of extracts were prepared and analyzed by Western blotting (WB) with anti-Aurora-A, anti-HURP, anti-cyclin B1, or anti-actin antibodies. Actin was used as a loading control. The similar cyclin B1 expression level indicates a comparable synchronization effect for pSUPER-Aurora-A transfectants during mitosis. The asterisk indicates the phosphorylation form of HURP.

    Techniques Used: Recombinant, Incubation, SDS Page, Autoradiography, Staining, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing

    Aurora-A phosphorylates HURP at four residues in vitro. (A) HURP-WT and various deletion constructs were subjected to in vitro transcription and translation in the presence of [ 35 S]methionine, followed by incubation with Aurora-A-WT or -KD, respectively. The phosphorylation status of HURPs was judged by mobility shift revealed by SDS-PAGE and autoradiography. (B) Recombinant HURPs were incubated with Aurora-A in kinase reaction buffer. After reaction at 30°C for 30 min, the samples were subjected to SDS-PAGE and stained with Coomassie blue. HURP bands were carefully sliced from the gel, and phosphorylation sites were determined by LC MS/MS analysis. Four residues were determined to be Aurora-A-dependent phosphorylation sites (the residues with asterisks above them in the upper four graphs) with 65% amino acid sequence coverage (the lower sequence; underlined amino acids represent those residues resolved by LC MS/MS). An asterisk indicates a phosphorylation site. (C) The four Aurora-A in vitro phosphorylation sites on HURP, which had been determined by LC MS/MS, were replaced with alanine (designated HURP-4P). 293T cells were transiently transfected with HA-HURP-WT or HA-HURP-4P. Twenty-four hours after transfection, the cells were lysed and immunoprecipitated (IP) with anti-HA antibody. Immunocomplexes were incubated with Aurora-A in the kinase reaction buffer in the presence of [γ- 32 P]ATP at 30°C for 30 min. Reaction mixtures were separated by SDS-PAGE and analyzed using Western blotting (WB) with anti-HA antibody (left) or autoradiography (right). The asterisk indicates the phosphorylation form of HURP. The HURP-4P mutant showed no [γ- 32 P]ATP incorporation. (D) 293T cells were transiently transfected with HA-HURP-WT or HA-HURP-4P. Twenty-four hours after transfection, the 293T cells were either left untreated (referred to as asynchronized) or synchronized in mitosis by nocodazole treatment for 16 h. Equal amounts of extracts were prepared and analyzed by Western blotting with anti-HA antibody. A mobility upshift band (indicated by the asterisk) was found for HA-HURP-WT, but not HA-HURP-4P, suggesting differences in protein phosphorylation. Similar results were obtained in two independent experiments.
    Figure Legend Snippet: Aurora-A phosphorylates HURP at four residues in vitro. (A) HURP-WT and various deletion constructs were subjected to in vitro transcription and translation in the presence of [ 35 S]methionine, followed by incubation with Aurora-A-WT or -KD, respectively. The phosphorylation status of HURPs was judged by mobility shift revealed by SDS-PAGE and autoradiography. (B) Recombinant HURPs were incubated with Aurora-A in kinase reaction buffer. After reaction at 30°C for 30 min, the samples were subjected to SDS-PAGE and stained with Coomassie blue. HURP bands were carefully sliced from the gel, and phosphorylation sites were determined by LC MS/MS analysis. Four residues were determined to be Aurora-A-dependent phosphorylation sites (the residues with asterisks above them in the upper four graphs) with 65% amino acid sequence coverage (the lower sequence; underlined amino acids represent those residues resolved by LC MS/MS). An asterisk indicates a phosphorylation site. (C) The four Aurora-A in vitro phosphorylation sites on HURP, which had been determined by LC MS/MS, were replaced with alanine (designated HURP-4P). 293T cells were transiently transfected with HA-HURP-WT or HA-HURP-4P. Twenty-four hours after transfection, the cells were lysed and immunoprecipitated (IP) with anti-HA antibody. Immunocomplexes were incubated with Aurora-A in the kinase reaction buffer in the presence of [γ- 32 P]ATP at 30°C for 30 min. Reaction mixtures were separated by SDS-PAGE and analyzed using Western blotting (WB) with anti-HA antibody (left) or autoradiography (right). The asterisk indicates the phosphorylation form of HURP. The HURP-4P mutant showed no [γ- 32 P]ATP incorporation. (D) 293T cells were transiently transfected with HA-HURP-WT or HA-HURP-4P. Twenty-four hours after transfection, the 293T cells were either left untreated (referred to as asynchronized) or synchronized in mitosis by nocodazole treatment for 16 h. Equal amounts of extracts were prepared and analyzed by Western blotting with anti-HA antibody. A mobility upshift band (indicated by the asterisk) was found for HA-HURP-WT, but not HA-HURP-4P, suggesting differences in protein phosphorylation. Similar results were obtained in two independent experiments.

    Techniques Used: In Vitro, Construct, Incubation, Mobility Shift, SDS Page, Autoradiography, Recombinant, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Transfection, Immunoprecipitation, Western Blot, Mutagenesis

    Related Articles

    Stable Transfection:

    Article Title: Mammalian Cdh1/Fzr mediates its own degradation
    Article Snippet: All the experiments, except those in , were performed with stably transfected lines to prevent fluctuations due to transfection efficiency. .. They were incubated overnight with the precipitate, washed the following morning, were allowed to recover for several hours and were treated with nocodazole for about 16 h. Nocodazole (Sigma—2 μ M ) and roscovitin (Biomol—28 μ M ) were dissolved in DMSO.

    Blocking Assay:

    Article Title: APC/CCdc20 targets E2F1 for degradation in prometaphase
    Article Snippet: Briefly, HeLa cells were treated with 2 mM thymidine (Sigma) for 19 h, washed with PBS and cultured in fresh media for 9 h. The cells were treated again with thymidine for 16 h and were released from the block by washing in PBS and replacing the media with fresh DMEM + 10% FBS. .. Synchronization at prometaphase with nocodazole was performed by incubating cells for 16 h with 330 nM nocodazole (Sigma).

    Article Title: Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry *
    Article Snippet: .. HeLa cells were synchronized to G2 /M phase by blocking with 2 m m thymidine (catalog no. T1895, Sigma) for 18 h, released in complete medium for 4 h, and then incubated in 100 ng/ml nocodazole (catalog no. M1404, Sigma) for 12 h. The HEK 293T, U2OS, and HCT116 cells were synchronized to G2 /M by incubation with 100 ng/ml nocodazole, taxol (catalog no. T7402, Sigma), or vinblastine (catalog no. V1377, Sigma) for 16 h. For G1 /S phase synchronization, HeLa cells were incubated with thymidine for 18 h, released for 9 h, and again incubated with thymidine for 18 h. G2 phase synchronization for HeLa and HEK 293T cells was achieved by incubation with 10 μ m RO-3306 (catalog no. 217699, Calbiochem) for 16 h. .. Asynchronous or mitotic cells were lysed in Laemmli sample buffer and subjected to Western blotting using the indicated antibodies.

    Incubation:

    Article Title: Elm1 kinase activates the spindle position checkpoint kinase Kin4
    Article Snippet: .. To arrest the cells with nocodazole, 15 µg/ml nocodazole (Sigma-Aldrich) was added to the culture media and incubated 2–4 h until > 90% of the cells arrested with large buds and one DNA-stained region (DAPI staining). .. S phase arrest was induced by adding 200 µM hydroxyurea (Sigma-Aldrich) to log phase cultures and further incubating for 2 h. To provide late anaphase arrest, 2% galactose was added to the log phase culture of Gal1-clb2ΔDB cells grown in YP-Raf medium.

    Article Title: Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis
    Article Snippet: .. To arrest the cells with nocodazole, 15 μg/mL nocodazole (Sigma-Aldrich) was added to the culture media and incubated 2–4 h, until > 90% of the cells arrested with large buds and one DNA-stained region (DAPI staining). ..

    Article Title: Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae
    Article Snippet: The cells were washed 10 times with PBS/BSA and incubated with secondary antibody (1:500 CY3-conjugated goat-antimouse or goat-antirabbit IgG) in PBS/BSA for 1 h at room temperature in a dark humidified chamber. .. 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.

    Article Title: Mammalian Cdh1/Fzr mediates its own degradation
    Article Snippet: .. They were incubated overnight with the precipitate, washed the following morning, were allowed to recover for several hours and were treated with nocodazole for about 16 h. Nocodazole (Sigma—2 μ M ) and roscovitin (Biomol—28 μ M ) were dissolved in DMSO. .. CAT assays were performed by standard methods ( ) using [14 C]chloramphenicol (Amersham) and acetyl CoA (Roche).

    Article Title: Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry *
    Article Snippet: .. HeLa cells were synchronized to G2 /M phase by blocking with 2 m m thymidine (catalog no. T1895, Sigma) for 18 h, released in complete medium for 4 h, and then incubated in 100 ng/ml nocodazole (catalog no. M1404, Sigma) for 12 h. The HEK 293T, U2OS, and HCT116 cells were synchronized to G2 /M by incubation with 100 ng/ml nocodazole, taxol (catalog no. T7402, Sigma), or vinblastine (catalog no. V1377, Sigma) for 16 h. For G1 /S phase synchronization, HeLa cells were incubated with thymidine for 18 h, released for 9 h, and again incubated with thymidine for 18 h. G2 phase synchronization for HeLa and HEK 293T cells was achieved by incubation with 10 μ m RO-3306 (catalog no. 217699, Calbiochem) for 16 h. .. Asynchronous or mitotic cells were lysed in Laemmli sample buffer and subjected to Western blotting using the indicated antibodies.

    Article Title: Characterizing Cell Adhesion by Using Micropipette Aspiration
    Article Snippet: .. When treated with nocodazole, cells were incubated for 1 h in culture medium containing 10 μ g/mL of nocodazole (Sigma-Aldrich).We verified using live-cell microtubule staining (tubulin tracker green, Life Technologies, Saint Aubin, France) that in these conditions, microtubules were properly destabilized. ..

    Activity Assay:

    Article Title: Mammalian Cdh1/Fzr mediates its own degradation
    Article Snippet: They were incubated overnight with the precipitate, washed the following morning, were allowed to recover for several hours and were treated with nocodazole for about 16 h. Nocodazole (Sigma—2 μ M ) and roscovitin (Biomol—28 μ M ) were dissolved in DMSO. .. The plotted CAT activity represents the proportion of di-acetylated [14 C]chloramphenicol of the total amount of [14 C]chloramphenicol, as quantified by a Fuji phosphorimager.

    Expressing:

    Article Title: Sumoylated BubR1 plays an important role in chromosome segregation and mitotic timing
    Article Snippet: HeLa cell line constitutively expressing His6 -SUMO-1 was kindly provided by Dr. Ronald Hay (University of Dundee). .. Mitotic shake-off cells were obtained from gentle tapping of either normally growing mitotic (rounded up) cells or cells treated with nocodazole (50 ng/mL) (Sigma-Aldrich) for 14 h. Both types of shake-off cells were used for mitotic release in the presence or absence of nocodazole (or taxol) and/or caffeine (3 mM, Sigma-Aldrich) as specified in each experiment.

    Article Title: Mammalian Cdh1/Fzr mediates its own degradation
    Article Snippet: The various CAT fusion expression vectors were co-transfected with a G418 resistance vector, and resistant colonies were pooled. .. They were incubated overnight with the precipitate, washed the following morning, were allowed to recover for several hours and were treated with nocodazole for about 16 h. Nocodazole (Sigma—2 μ M ) and roscovitin (Biomol—28 μ M ) were dissolved in DMSO.

    Article Title: Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration
    Article Snippet: Intracerebroventricular cannulated (ICVC) C57BL/6BomTac mice (Taconic), males aged 10 weeks, received 6-μl infusions of either nocodazole (30 nM in 20% Captisol; CyDex Pharmaceuticals) or nocodazole with noscapine (10 nM; Sigma-Aldrich) and nocodazole with taxol (10 nM; Sigma-Aldrich) over a period of 6 minutes. .. In the studies with mice expressing ALS-linked mutant SOD1G93A , noscapine (Sigma-Aldrich) was injected intraperitoneally at 200 mg/kg/d, and riluzole was given in diet, with the final drug dose computed to be 44 mg/kg/d.

    Modification:

    Article Title: Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy
    Article Snippet: The prevalence of posttranslationally modified α-tubulin in cardiocyte microtubules was used as the primary measure of the age, and thus the intrinsic stability, of these polymers. .. For this purpose, freshly isolated cardiocytes were sedimented onto laminin-coated coverslips at 1 g for 45 min, washed twice with 0.3 μM nocodazole ( Aldrich Chemical Co. , Milwaukee, WI) in PBS, exposed to 0.3 μM nocodazole in M-199 medium for 0.0, 0.5, 1.0, or 1.5 h, extracted for 1 min in 1% Triton X-100 ( Sigma ) in the microtubule-stabilizing buffer, all at 25°C, and prepared as above for indirect immunofluorescence confocal microscopy using the antibody to β-tubulin (DM1B; Amersham ).

    Article Title: APC/CCdc20 targets E2F1 for degradation in prometaphase
    Article Snippet: HeLa cervical carcinoma cells and T98G glioblastoma cells obtained from ATCC were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% FBS at 37°C with 5% CO2 . .. Synchronization at prometaphase with nocodazole was performed by incubating cells for 16 h with 330 nM nocodazole (Sigma).

    Article Title: Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry *
    Article Snippet: Human cervical carcinoma HeLa, osteosarcoma U2OS, colorectal carcinoma HCT116, and embryonic kidney HEK 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin, and 2 m m l -glutamine at 37 °C with 5% CO2 (hereafter referred to as complete medium). .. HeLa cells were synchronized to G2 /M phase by blocking with 2 m m thymidine (catalog no. T1895, Sigma) for 18 h, released in complete medium for 4 h, and then incubated in 100 ng/ml nocodazole (catalog no. M1404, Sigma) for 12 h. The HEK 293T, U2OS, and HCT116 cells were synchronized to G2 /M by incubation with 100 ng/ml nocodazole, taxol (catalog no. T7402, Sigma), or vinblastine (catalog no. V1377, Sigma) for 16 h. For G1 /S phase synchronization, HeLa cells were incubated with thymidine for 18 h, released for 9 h, and again incubated with thymidine for 18 h. G2 phase synchronization for HeLa and HEK 293T cells was achieved by incubation with 10 μ m RO-3306 (catalog no. 217699, Calbiochem) for 16 h.

    Transfection:

    Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1
    Article Snippet: Cycloheximide (CHX) (CAT#C1988), MG132 (CAT#C2211), Thymidine (CAT # T9250), Micrococcal nuclease (MNase) (CAT# N3755) and Schneider’s Insect Medium (CAT#S0146) were from Millipore SIGMA and polyethylenimine (PEI) (CAT#23966-1) was from Polysciences Inc. N -methylmaleimide (NEM) (CAT#ETM222), was from Bioshop, CDK1 inhibitor RO-3306 (10 μM) (CAT# 217699) and Nocodazole (CAT#487928) where purchased from Millipore. .. Effectene transfection kit (CAT#301427) was from Qiagen.

    Article Title: Assembling an intermediate filament network by dynamic cotranslation
    Article Snippet: .. For live imaging of cells in nocodazole (whole cell FRAP experiments and observation of particle formation experiments), PC12 cells were transfected with the appropriate constructs for 24 h, differentiated in NGF for 2–4 h, and then treated with 10 μg/ml nocodazole (Sigma-Aldrich) for 30 min before imaging. .. For analysis of mRNA clustering in nocodazole, cells were transfected with the ECFP-peripherin-R24-3′UTR construct for 24 h and then treated with both NGF and nocodazole for 4 h before quantitative FISH analysis.

    Article Title: Mammalian Cdh1/Fzr mediates its own degradation
    Article Snippet: Paragraph title: Cell culture, transfections, synchronization and CAT assays ... They were incubated overnight with the precipitate, washed the following morning, were allowed to recover for several hours and were treated with nocodazole for about 16 h. Nocodazole (Sigma—2 μ M ) and roscovitin (Biomol—28 μ M ) were dissolved in DMSO.

    Cell Culture:

    Article Title: Elm1 kinase activates the spindle position checkpoint kinase Kin4
    Article Snippet: Paragraph title: Cell culture synchronizations ... To arrest the cells with nocodazole, 15 µg/ml nocodazole (Sigma-Aldrich) was added to the culture media and incubated 2–4 h until > 90% of the cells arrested with large buds and one DNA-stained region (DAPI staining).

    Article Title: APC/CCdc20 targets E2F1 for degradation in prometaphase
    Article Snippet: Briefly, HeLa cells were treated with 2 mM thymidine (Sigma) for 19 h, washed with PBS and cultured in fresh media for 9 h. The cells were treated again with thymidine for 16 h and were released from the block by washing in PBS and replacing the media with fresh DMEM + 10% FBS. .. Synchronization at prometaphase with nocodazole was performed by incubating cells for 16 h with 330 nM nocodazole (Sigma).

    Article Title: Assembling an intermediate filament network by dynamic cotranslation
    Article Snippet: Paragraph title: Cell culture ... For live imaging of cells in nocodazole (whole cell FRAP experiments and observation of particle formation experiments), PC12 cells were transfected with the appropriate constructs for 24 h, differentiated in NGF for 2–4 h, and then treated with 10 μg/ml nocodazole (Sigma-Aldrich) for 30 min before imaging.

    Article Title: Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis
    Article Snippet: Paragraph title: Cell culture synchronization ... To arrest the cells with nocodazole, 15 μg/mL nocodazole (Sigma-Aldrich) was added to the culture media and incubated 2–4 h, until > 90% of the cells arrested with large buds and one DNA-stained region (DAPI staining).

    Article Title: Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae
    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. .. After a 3-h incubation at 25°C, > 80% of the nocodazole-treated cells contained a large bud and a single unelongated nucleus in the mother cell.

    Article Title: Sumoylated BubR1 plays an important role in chromosome segregation and mitotic timing
    Article Snippet: Paragraph title: Cell culture. ... Mitotic shake-off cells were obtained from gentle tapping of either normally growing mitotic (rounded up) cells or cells treated with nocodazole (50 ng/mL) (Sigma-Aldrich) for 14 h. Both types of shake-off cells were used for mitotic release in the presence or absence of nocodazole (or taxol) and/or caffeine (3 mM, Sigma-Aldrich) as specified in each experiment.

    Article Title: Mammalian Cdh1/Fzr mediates its own degradation
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    Article Title: Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry *
    Article Snippet: Paragraph title: Cell Culture and Synchronization ... HeLa cells were synchronized to G2 /M phase by blocking with 2 m m thymidine (catalog no. T1895, Sigma) for 18 h, released in complete medium for 4 h, and then incubated in 100 ng/ml nocodazole (catalog no. M1404, Sigma) for 12 h. The HEK 293T, U2OS, and HCT116 cells were synchronized to G2 /M by incubation with 100 ng/ml nocodazole, taxol (catalog no. T7402, Sigma), or vinblastine (catalog no. V1377, Sigma) for 16 h. For G1 /S phase synchronization, HeLa cells were incubated with thymidine for 18 h, released for 9 h, and again incubated with thymidine for 18 h. G2 phase synchronization for HeLa and HEK 293T cells was achieved by incubation with 10 μ m RO-3306 (catalog no. 217699, Calbiochem) for 16 h.

    Article Title: Characterizing Cell Adhesion by Using Micropipette Aspiration
    Article Snippet: Before any experiment, a μ -Dish was brought onto the stage of the microscope, where experiments were performed, at room temperature in cultured medium with 20 mM HEPES (Invitrogen) added, on cells that had been plated onto the petri dish a few hours to a few days earlier. .. When treated with nocodazole, cells were incubated for 1 h in culture medium containing 10 μ g/mL of nocodazole (Sigma-Aldrich).We verified using live-cell microtubule staining (tubulin tracker green, Life Technologies, Saint Aubin, France) that in these conditions, microtubules were properly destabilized.

    Inhibition:

    Article Title: Assembling an intermediate filament network by dynamic cotranslation
    Article Snippet: For live imaging of cells in nocodazole (whole cell FRAP experiments and observation of particle formation experiments), PC12 cells were transfected with the appropriate constructs for 24 h, differentiated in NGF for 2–4 h, and then treated with 10 μg/ml nocodazole (Sigma-Aldrich) for 30 min before imaging. .. For translation inhibition experiments, cells were treated with either 10 μg/ml cycloheximide (Calbiochem) or 10 μg/ml puromycin (Sigma-Aldrich) for time periods of 5 min to 1 h. Rat2 cells were maintained in DME containing 10% fetal calf serum at 37°C.

    Imaging:

    Article Title: Assembling an intermediate filament network by dynamic cotranslation
    Article Snippet: .. For live imaging of cells in nocodazole (whole cell FRAP experiments and observation of particle formation experiments), PC12 cells were transfected with the appropriate constructs for 24 h, differentiated in NGF for 2–4 h, and then treated with 10 μg/ml nocodazole (Sigma-Aldrich) for 30 min before imaging. .. For analysis of mRNA clustering in nocodazole, cells were transfected with the ECFP-peripherin-R24-3′UTR construct for 24 h and then treated with both NGF and nocodazole for 4 h before quantitative FISH analysis.

    Injection:

    Article Title: Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration
    Article Snippet: Intracerebroventricular cannulated (ICVC) C57BL/6BomTac mice (Taconic), males aged 10 weeks, received 6-μl infusions of either nocodazole (30 nM in 20% Captisol; CyDex Pharmaceuticals) or nocodazole with noscapine (10 nM; Sigma-Aldrich) and nocodazole with taxol (10 nM; Sigma-Aldrich) over a period of 6 minutes. .. In the studies with mice expressing ALS-linked mutant SOD1G93A , noscapine (Sigma-Aldrich) was injected intraperitoneally at 200 mg/kg/d, and riluzole was given in diet, with the final drug dose computed to be 44 mg/kg/d.

    Immunofluorescence:

    Article Title: Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy
    Article Snippet: .. For this purpose, freshly isolated cardiocytes were sedimented onto laminin-coated coverslips at 1 g for 45 min, washed twice with 0.3 μM nocodazole ( Aldrich Chemical Co. , Milwaukee, WI) in PBS, exposed to 0.3 μM nocodazole in M-199 medium for 0.0, 0.5, 1.0, or 1.5 h, extracted for 1 min in 1% Triton X-100 ( Sigma ) in the microtubule-stabilizing buffer, all at 25°C, and prepared as above for indirect immunofluorescence confocal microscopy using the antibody to β-tubulin (DM1B; Amersham ). ..

    Article Title: Assembling an intermediate filament network by dynamic cotranslation
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    Article Title: Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae
    Article Snippet: Paragraph title: Immunofluorescence Microscopy ... 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.

    Mutagenesis:

    Article Title: Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae
    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. .. Vacuoles in wild-type and aux1/swa2 mutant cells were labeled with 80 μM FM 4-64 (Molecular Probes, Eugene, OR) according to the method of .

    Article Title: Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration
    Article Snippet: Transgenic mice carrying the human A53T mutant SNCA cDNA driven by the mouse prion protein promoter homozygosed on a Snca -null background [ Sncatm1Nbm ;Tg( Prnp-SNCA*A53T ) mice] ( ) were bred in house at the National Genome Research Institute/NIH (NHGRI/NIH) facility. .. Intracerebroventricular cannulated (ICVC) C57BL/6BomTac mice (Taconic), males aged 10 weeks, received 6-μl infusions of either nocodazole (30 nM in 20% Captisol; CyDex Pharmaceuticals) or nocodazole with noscapine (10 nM; Sigma-Aldrich) and nocodazole with taxol (10 nM; Sigma-Aldrich) over a period of 6 minutes.

    Isolation:

    Article Title: Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy
    Article Snippet: .. For this purpose, freshly isolated cardiocytes were sedimented onto laminin-coated coverslips at 1 g for 45 min, washed twice with 0.3 μM nocodazole ( Aldrich Chemical Co. , Milwaukee, WI) in PBS, exposed to 0.3 μM nocodazole in M-199 medium for 0.0, 0.5, 1.0, or 1.5 h, extracted for 1 min in 1% Triton X-100 ( Sigma ) in the microtubule-stabilizing buffer, all at 25°C, and prepared as above for indirect immunofluorescence confocal microscopy using the antibody to β-tubulin (DM1B; Amersham ). ..

    Microscopy:

    Article Title: Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae
    Article Snippet: Paragraph title: Immunofluorescence Microscopy ... 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.

    Article Title: Characterizing Cell Adhesion by Using Micropipette Aspiration
    Article Snippet: Before any experiment, a μ -Dish was brought onto the stage of the microscope, where experiments were performed, at room temperature in cultured medium with 20 mM HEPES (Invitrogen) added, on cells that had been plated onto the petri dish a few hours to a few days earlier. .. When treated with nocodazole, cells were incubated for 1 h in culture medium containing 10 μ g/mL of nocodazole (Sigma-Aldrich).We verified using live-cell microtubule staining (tubulin tracker green, Life Technologies, Saint Aubin, France) that in these conditions, microtubules were properly destabilized.

    Mouse Assay:

    Article Title: Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration
    Article Snippet: .. Intracerebroventricular cannulated (ICVC) C57BL/6BomTac mice (Taconic), males aged 10 weeks, received 6-μl infusions of either nocodazole (30 nM in 20% Captisol; CyDex Pharmaceuticals) or nocodazole with noscapine (10 nM; Sigma-Aldrich) and nocodazole with taxol (10 nM; Sigma-Aldrich) over a period of 6 minutes. ..

    Transgenic Assay:

    Article Title: Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration
    Article Snippet: Transgenic mice carrying the human A53T mutant SNCA cDNA driven by the mouse prion protein promoter homozygosed on a Snca -null background [ Sncatm1Nbm ;Tg( Prnp-SNCA*A53T ) mice] ( ) were bred in house at the National Genome Research Institute/NIH (NHGRI/NIH) facility. .. Intracerebroventricular cannulated (ICVC) C57BL/6BomTac mice (Taconic), males aged 10 weeks, received 6-μl infusions of either nocodazole (30 nM in 20% Captisol; CyDex Pharmaceuticals) or nocodazole with noscapine (10 nM; Sigma-Aldrich) and nocodazole with taxol (10 nM; Sigma-Aldrich) over a period of 6 minutes.

    Labeling:

    Article Title: Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae
    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. .. Vacuoles in wild-type and aux1/swa2 mutant cells were labeled with 80 μM FM 4-64 (Molecular Probes, Eugene, OR) according to the method of .

    Article Title: Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration
    Article Snippet: Paragraph title: Animal studies and 2 H2 O labeling. ... Intracerebroventricular cannulated (ICVC) C57BL/6BomTac mice (Taconic), males aged 10 weeks, received 6-μl infusions of either nocodazole (30 nM in 20% Captisol; CyDex Pharmaceuticals) or nocodazole with noscapine (10 nM; Sigma-Aldrich) and nocodazole with taxol (10 nM; Sigma-Aldrich) over a period of 6 minutes.

    Confocal Microscopy:

    Article Title: Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy
    Article Snippet: .. For this purpose, freshly isolated cardiocytes were sedimented onto laminin-coated coverslips at 1 g for 45 min, washed twice with 0.3 μM nocodazole ( Aldrich Chemical Co. , Milwaukee, WI) in PBS, exposed to 0.3 μM nocodazole in M-199 medium for 0.0, 0.5, 1.0, or 1.5 h, extracted for 1 min in 1% Triton X-100 ( Sigma ) in the microtubule-stabilizing buffer, all at 25°C, and prepared as above for indirect immunofluorescence confocal microscopy using the antibody to β-tubulin (DM1B; Amersham ). ..

    Staining:

    Article Title: Elm1 kinase activates the spindle position checkpoint kinase Kin4
    Article Snippet: .. To arrest the cells with nocodazole, 15 µg/ml nocodazole (Sigma-Aldrich) was added to the culture media and incubated 2–4 h until > 90% of the cells arrested with large buds and one DNA-stained region (DAPI staining). .. S phase arrest was induced by adding 200 µM hydroxyurea (Sigma-Aldrich) to log phase cultures and further incubating for 2 h. To provide late anaphase arrest, 2% galactose was added to the log phase culture of Gal1-clb2ΔDB cells grown in YP-Raf medium.

    Article Title: Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis
    Article Snippet: .. To arrest the cells with nocodazole, 15 μg/mL nocodazole (Sigma-Aldrich) was added to the culture media and incubated 2–4 h, until > 90% of the cells arrested with large buds and one DNA-stained region (DAPI staining). ..

    Article Title: Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae
    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. .. The cells were fixed as described above and nuclei were stained by resuspending the fixed cells in mounting medium containing 25 ng/ml DAPI.

    Article Title: Characterizing Cell Adhesion by Using Micropipette Aspiration
    Article Snippet: .. When treated with nocodazole, cells were incubated for 1 h in culture medium containing 10 μ g/mL of nocodazole (Sigma-Aldrich).We verified using live-cell microtubule staining (tubulin tracker green, Life Technologies, Saint Aubin, France) that in these conditions, microtubules were properly destabilized. ..

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1
    Article Snippet: .. Cycloheximide (CHX) (CATC1988), MG132 (CAT#C2211), Thymidine (CAT # T9250), Micrococcal nuclease (MNase) (CAT# N3755) and Schneider’s Insect Medium (CAT#S0146) were from Millipore SIGMA and polyethylenimine (PEI) (CAT#23966-1) was from Polysciences Inc. N -methylmaleimide (NEM) (CAT#ETM222), was from Bioshop, CDK1 inhibitor RO-3306 (10 μM) (CAT# 217699) and Nocodazole (CAT#487928) where purchased from Millipore. .. Effectene transfection kit (CAT#301427) was from Qiagen.

    Article Title: Mammalian Cdh1/Fzr mediates its own degradation
    Article Snippet: Paragraph title: Cell culture, transfections, synchronization and CAT assays ... They were incubated overnight with the precipitate, washed the following morning, were allowed to recover for several hours and were treated with nocodazole for about 16 h. Nocodazole (Sigma—2 μ M ) and roscovitin (Biomol—28 μ M ) were dissolved in DMSO.

    Chromatin Immunoprecipitation:

    Article Title: Misregulation of Scm3p/HJURP Causes Chromosome Instability in Saccharomyces cerevisiae and Human Cells
    Article Snippet: Samples were taken at 15 min time points after release from G1 arrest and used for FACS, protein and ChIP analysis. .. Additional synchronizations were carried out to confirm the results: cells were arrested in G1 with α-factor as described above and released into YPD medium containing 15 µg/ml nocodazole (M1404, Sigma); cells were arrested in G2/M with nocodazole for 2 hours and released into YPD medium containing 3 µM α-factor (T-6901, Sigma).

    Plasmid Preparation:

    Article Title: Mammalian Cdh1/Fzr mediates its own degradation
    Article Snippet: The various CAT fusion expression vectors were co-transfected with a G418 resistance vector, and resistant colonies were pooled. .. They were incubated overnight with the precipitate, washed the following morning, were allowed to recover for several hours and were treated with nocodazole for about 16 h. Nocodazole (Sigma—2 μ M ) and roscovitin (Biomol—28 μ M ) were dissolved in DMSO.

    Concentration Assay:

    Article Title: The Temperature-Sensitive Role of Cryptococcus neoformansROM2 in Cell Morphogenesis
    Article Snippet: .. Nocodazole Ten fold serial dilutions from 104 to 101 of KN99α, KN99α rom2 and KN99α rom2 +ROM2 cells were plated in a volume of 5 µL on YPD containing 0, 0.1 µM, or 0.2 µM nocodazole (Sigma) from a stock concentration of 100 µg ml−1 nocodazole dissolved in DMSO. ..

    Construct:

    Article Title: Assembling an intermediate filament network by dynamic cotranslation
    Article Snippet: .. For live imaging of cells in nocodazole (whole cell FRAP experiments and observation of particle formation experiments), PC12 cells were transfected with the appropriate constructs for 24 h, differentiated in NGF for 2–4 h, and then treated with 10 μg/ml nocodazole (Sigma-Aldrich) for 30 min before imaging. .. For analysis of mRNA clustering in nocodazole, cells were transfected with the ECFP-peripherin-R24-3′UTR construct for 24 h and then treated with both NGF and nocodazole for 4 h before quantitative FISH analysis.

    FACS:

    Article Title: Misregulation of Scm3p/HJURP Causes Chromosome Instability in Saccharomyces cerevisiae and Human Cells
    Article Snippet: Samples were taken at 15 min time points after release from G1 arrest and used for FACS, protein and ChIP analysis. .. Additional synchronizations were carried out to confirm the results: cells were arrested in G1 with α-factor as described above and released into YPD medium containing 15 µg/ml nocodazole (M1404, Sigma); cells were arrested in G2/M with nocodazole for 2 hours and released into YPD medium containing 3 µM α-factor (T-6901, Sigma).

    Fluorescence In Situ Hybridization:

    Article Title: Assembling an intermediate filament network by dynamic cotranslation
    Article Snippet: For live imaging of cells in nocodazole (whole cell FRAP experiments and observation of particle formation experiments), PC12 cells were transfected with the appropriate constructs for 24 h, differentiated in NGF for 2–4 h, and then treated with 10 μg/ml nocodazole (Sigma-Aldrich) for 30 min before imaging. .. For analysis of mRNA clustering in nocodazole, cells were transfected with the ECFP-peripherin-R24-3′UTR construct for 24 h and then treated with both NGF and nocodazole for 4 h before quantitative FISH analysis.

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    Millipore nocodazole
    G601S- and F805C-Kv11.1 proteins show distinct immunostaining patterns. Shown are representative confocal images of HEK293 cells expressing (A) G601S- or (B) F805C-Kv11.1 protein immunostained with anti-Kv11.1 (red, first column), anti-Bap31 (green, second column) in control conditions (top row; n = 13 images for G601S and n = 17 images for F805C) or after incubation in <t>nocodazole</t> (bottom row; n = 15 images for G601S and n = 16 images for F805C). The overlay images are also shown (overlapping immunostaining is yellow, third column), and the white dashed box portion of the overlay image is shown in larger detail (fourth column). The nuclei are labeled blue. The arrowheads in (A) highlight the reticular immunostaining pattern of G601S-Kv11.1 protein after nocodazole treatment, the arrowheads in (B) highlight the unique immunostaining pattern of F805C-Kv11.1 protein, and the scale bar represents 10 μm. Two independent cultures were tested for each condition.
    Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    Millipore nocodazole containing medium
    CDK6 and CDC25A are downstream effectors of NANOG. (A) Quantitative RT-PCR analysis showing down-regulation of CDK6 and CDC25A in H1 NANOG, H9 NANOG, and hES-NCL1 NANOG sublines 42 h after the transfection of CDK6 and CDC25A siRNA. The data represent the mean ± SEM (error bars) from three experiments (one in each subline). The value for the control-transfected sample (vector only) was set to 1, and all other values were calculated with respect to this. (B) Western blotting showing down-regulation of CDK6 and CDC25A in H1 NANOG sublines. Molecular masses are indicated in kilodaltons. (C) Chart representation of the fraction of cells in S phase over time after transfection of CDK6 siRNA and synchronization by <t>nocodazole</t> for 18 h in the H9 NANOG subline assessed by propidium iodide staining. This figure represents an example of at least two independent experiments carried in the H9 subline. (D) Flow cytometry images showing retention of cells in G1 phase of the cell cycle after transfection of CDC25A and NANOG siRNAs and synchronization by nocodazole for 18 h in the H9 NANOG subline assessed by propidium iodide staining. This figure represents an example of at least two independent experiments performed in the H9 subline.
    Nocodazole Containing Medium, 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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    Image Search Results


    G601S- and F805C-Kv11.1 proteins show distinct immunostaining patterns. Shown are representative confocal images of HEK293 cells expressing (A) G601S- or (B) F805C-Kv11.1 protein immunostained with anti-Kv11.1 (red, first column), anti-Bap31 (green, second column) in control conditions (top row; n = 13 images for G601S and n = 17 images for F805C) or after incubation in nocodazole (bottom row; n = 15 images for G601S and n = 16 images for F805C). The overlay images are also shown (overlapping immunostaining is yellow, third column), and the white dashed box portion of the overlay image is shown in larger detail (fourth column). The nuclei are labeled blue. The arrowheads in (A) highlight the reticular immunostaining pattern of G601S-Kv11.1 protein after nocodazole treatment, the arrowheads in (B) highlight the unique immunostaining pattern of F805C-Kv11.1 protein, and the scale bar represents 10 μm. Two independent cultures were tested for each condition.

    Journal: Frontiers in Physiology

    Article Title: Visualizing Mutation-Specific Differences in the Trafficking-Deficient Phenotype of Kv11.1 Proteins Linked to Long QT Syndrome Type 2

    doi: 10.3389/fphys.2018.00584

    Figure Lengend Snippet: G601S- and F805C-Kv11.1 proteins show distinct immunostaining patterns. Shown are representative confocal images of HEK293 cells expressing (A) G601S- or (B) F805C-Kv11.1 protein immunostained with anti-Kv11.1 (red, first column), anti-Bap31 (green, second column) in control conditions (top row; n = 13 images for G601S and n = 17 images for F805C) or after incubation in nocodazole (bottom row; n = 15 images for G601S and n = 16 images for F805C). The overlay images are also shown (overlapping immunostaining is yellow, third column), and the white dashed box portion of the overlay image is shown in larger detail (fourth column). The nuclei are labeled blue. The arrowheads in (A) highlight the reticular immunostaining pattern of G601S-Kv11.1 protein after nocodazole treatment, the arrowheads in (B) highlight the unique immunostaining pattern of F805C-Kv11.1 protein, and the scale bar represents 10 μm. Two independent cultures were tested for each condition.

    Article Snippet: For pharmacological studies, we incubated cells in nocodazole (20 μM) (Millipore, Sigma, St. Louis, MO, United States), bortezomib, lactacystin, and MG132 (Millipore, Sigma, St. Louis, MO, United States), or E-4031 (10 μM) (Alomone Laboratories, Jerusalem, Israel).

    Techniques: Immunostaining, Expressing, Incubation, Labeling

    Involvement of microtubules in the transport of CPV to the perinuclear area. (A to C) Confocal live-cell imaging of SFV-infected cells, either untreated or in the presence of nocodazole (from the beginning of infection). BHK cells were infected with SFV-ZsG VRPs (MOI, 500), and imaging was performed at 4 h p.i. LysoTracker (red) was used to stain the acidic organelles. (A) (Left) Untreated sample, showing the late phenotype and the ZsG signal around the acidic organelles. (Right) Isosurface representation of the area boxed on the left. CPV were cut in half to illustrate the ZsG signal surrounding the LysoTracker staining. (Inset) Intact CPV in the boxed area, hiding the LysoTracker signal. (B) (Left) In the presence of nocodazole, RC-containing vesicles stay scattered. They are not transported to the perinuclear area, and only some ZsG signal colocalizes with LysoTracker as small patches on the surfaces of acidic organelles. The area boxed with solid lines was chosen for isosurface representation, while the area boxed with dashed lines was used for tracking of the RCs in panel C. Bar, 10 μm. (Right) Isosurface representation. Bar, 1 μm. (C) Tracking of the RCs in a nocodazole-treated sample. The rate of the recording was 0.5 frame/s (0.5 Hz). The total recording time was 1 min 36 s. Representative tracks are shown; statistics for one track (circled) are given on the right. Bar, 5 μm. (D) Nocodazole treatment does not interfere with the replication of SFV. BHK cells were infected with SFV (MOI, 500) in the presence of nocodazole, and cells were fixed at 4 h p.i. The scattered nsP3-positive vesicles (green) are also positive for dsRNA staining (red). Bars, 10 μm.

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes ▿Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes ▿ †

    doi: 10.1128/JVI.00477-10

    Figure Lengend Snippet: Involvement of microtubules in the transport of CPV to the perinuclear area. (A to C) Confocal live-cell imaging of SFV-infected cells, either untreated or in the presence of nocodazole (from the beginning of infection). BHK cells were infected with SFV-ZsG VRPs (MOI, 500), and imaging was performed at 4 h p.i. LysoTracker (red) was used to stain the acidic organelles. (A) (Left) Untreated sample, showing the late phenotype and the ZsG signal around the acidic organelles. (Right) Isosurface representation of the area boxed on the left. CPV were cut in half to illustrate the ZsG signal surrounding the LysoTracker staining. (Inset) Intact CPV in the boxed area, hiding the LysoTracker signal. (B) (Left) In the presence of nocodazole, RC-containing vesicles stay scattered. They are not transported to the perinuclear area, and only some ZsG signal colocalizes with LysoTracker as small patches on the surfaces of acidic organelles. The area boxed with solid lines was chosen for isosurface representation, while the area boxed with dashed lines was used for tracking of the RCs in panel C. Bar, 10 μm. (Right) Isosurface representation. Bar, 1 μm. (C) Tracking of the RCs in a nocodazole-treated sample. The rate of the recording was 0.5 frame/s (0.5 Hz). The total recording time was 1 min 36 s. Representative tracks are shown; statistics for one track (circled) are given on the right. Bar, 5 μm. (D) Nocodazole treatment does not interfere with the replication of SFV. BHK cells were infected with SFV (MOI, 500) in the presence of nocodazole, and cells were fixed at 4 h p.i. The scattered nsP3-positive vesicles (green) are also positive for dsRNA staining (red). Bars, 10 μm.

    Article Snippet: Nocodazole for tubulin disruption was obtained from Calbiochem (Merck KGaA, Germany).

    Techniques: Live Cell Imaging, Infection, Imaging, Staining

    Model for the trafficking of alphavirus RCs and the biogenesis of CPV-I. After SFV entry, which occurs via clathrin-mediated endocytosis, low-pH-triggered fusion releases the nucleocapsids, and viral mRNA is translated into a polyprotein. Protein-RNA complexes are transported to the PM by an unknown mechanism (white arrows). At the PM, spherule structures are formed (step 1), followed by endocytosis in a PI3K (inhibition by wortmannin)- and actin-myosin (inhibition by blebbistatin)-dependent manner. Small internalized vesicles carry a few spherules (step 2), and many homotypic fusions, as well as fusions with late endosomes, occur (step 3 [inhibition by nocodazole]). These larger, acidic, RC-containing vesicles are transported to the perinuclear area by using microtubules (inhibition by nocodazole), where the maturation of stable large CPV-I is completed (step 4). The acidic nature of the late vesicles is indicated by pink coloring. wort., wortmannin; EE, early endosome; LE, late endosome. Representative EM images from each step of the RC trafficking are shown to the right of the model.

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes ▿Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes ▿ †

    doi: 10.1128/JVI.00477-10

    Figure Lengend Snippet: Model for the trafficking of alphavirus RCs and the biogenesis of CPV-I. After SFV entry, which occurs via clathrin-mediated endocytosis, low-pH-triggered fusion releases the nucleocapsids, and viral mRNA is translated into a polyprotein. Protein-RNA complexes are transported to the PM by an unknown mechanism (white arrows). At the PM, spherule structures are formed (step 1), followed by endocytosis in a PI3K (inhibition by wortmannin)- and actin-myosin (inhibition by blebbistatin)-dependent manner. Small internalized vesicles carry a few spherules (step 2), and many homotypic fusions, as well as fusions with late endosomes, occur (step 3 [inhibition by nocodazole]). These larger, acidic, RC-containing vesicles are transported to the perinuclear area by using microtubules (inhibition by nocodazole), where the maturation of stable large CPV-I is completed (step 4). The acidic nature of the late vesicles is indicated by pink coloring. wort., wortmannin; EE, early endosome; LE, late endosome. Representative EM images from each step of the RC trafficking are shown to the right of the model.

    Article Snippet: Nocodazole for tubulin disruption was obtained from Calbiochem (Merck KGaA, Germany).

    Techniques: Inhibition

    Virus replication in the presence of inhibitors. One step growth curves of SFV-ZsG in the presence of wortmannin or nocodazole are shown. Released viruses were withdrawn at 2-h intervals as indicated, and virus titers were measured by direct fluorescence (see Materials and Methods). (Inset) Effects of the same inhibitors on SFV RNA synthesis. Infected BHK cells were pulse-labeled with [ 3 H]uridine at the indicated times, and labeled RNA was measured by scintillation counting after TCA precipitation. The results are averages for two independent experiments; error bars represent SDs.

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes ▿Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes ▿ †

    doi: 10.1128/JVI.00477-10

    Figure Lengend Snippet: Virus replication in the presence of inhibitors. One step growth curves of SFV-ZsG in the presence of wortmannin or nocodazole are shown. Released viruses were withdrawn at 2-h intervals as indicated, and virus titers were measured by direct fluorescence (see Materials and Methods). (Inset) Effects of the same inhibitors on SFV RNA synthesis. Infected BHK cells were pulse-labeled with [ 3 H]uridine at the indicated times, and labeled RNA was measured by scintillation counting after TCA precipitation. The results are averages for two independent experiments; error bars represent SDs.

    Article Snippet: Nocodazole for tubulin disruption was obtained from Calbiochem (Merck KGaA, Germany).

    Techniques: Fluorescence, Infection, Labeling, TCA Precipitation

    Fkh1 instability occurs specifically in mitosis ( A ) WT cells expressing endogenous FKH1-TAP were grown to mid log. CHX was added to the asynchronous cells to stop further protein synthesis. Samples were removed every 30 minutes and assessed by Western analyses with the antibodies shown. ( B ) The experiment described in ( A ) was performed, except the cells were arrested in G1 using α-factor. ( C ) The experiment described in ( A ) was performed, except the cells were arrested in mitosis using nocodazole. ( D ) The experiment described in ( A ) was performed, except the cells were arrested in S phase using hydroxyurea. (E) Blots from 3 repeat experiments of ( A ), ( B ), and ( C ), and 2 repeats of ( D ), were imaged with Fkh1-TAP levels compared over time to visualize Fkh1 stability. Standard error of the mean is shown. Samples were also removed following cell treatment for flow cytometry to verify cell cycle arrest. ( F ) apc10Δ Fkh1-TAP cells were arrested in G1 using α-factor. CHX was then added with samples removed every 30 minutes for protein analyses.

    Journal: Aging (Albany NY)

    Article Title: Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance

    doi: 10.18632/aging.100949

    Figure Lengend Snippet: Fkh1 instability occurs specifically in mitosis ( A ) WT cells expressing endogenous FKH1-TAP were grown to mid log. CHX was added to the asynchronous cells to stop further protein synthesis. Samples were removed every 30 minutes and assessed by Western analyses with the antibodies shown. ( B ) The experiment described in ( A ) was performed, except the cells were arrested in G1 using α-factor. ( C ) The experiment described in ( A ) was performed, except the cells were arrested in mitosis using nocodazole. ( D ) The experiment described in ( A ) was performed, except the cells were arrested in S phase using hydroxyurea. (E) Blots from 3 repeat experiments of ( A ), ( B ), and ( C ), and 2 repeats of ( D ), were imaged with Fkh1-TAP levels compared over time to visualize Fkh1 stability. Standard error of the mean is shown. Samples were also removed following cell treatment for flow cytometry to verify cell cycle arrest. ( F ) apc10Δ Fkh1-TAP cells were arrested in G1 using α-factor. CHX was then added with samples removed every 30 minutes for protein analyses.

    Article Snippet: For mitotic arrest, Nocodazole (Calbiochem) was used for approximately 3 hours, with 5 μg/ml added initially and then an additional 2.5 μg/ml added after 90 minutes.

    Techniques: Expressing, Western Blot, Flow Cytometry, Cytometry

    Mutation of the highly conserved K373 in Fkh1 stabilizes the protein in mitosis ( A ) Sequence alignment of Fkh1-like proteins across evolutionary boundaries highlighting the highly conserved lysine (K373). The mutation made is shown above in red. ( B ) Morphology of fkh2Δ cells harboring the FKH1 K-R mutation shown in ( A ). (C) fkh2Δ cells expressing FKH1 K-R were arrested in mitosis using nocodazole, then treated with CHX for 60 minutes. Proteins were then harvested and examined for Fkh1-TAP, Clb2 and GAPDH content. ( D ) All bands in ( C ) were imaged from three separate experiments and the ratio of intensities before and after CHX addition were determined. Standard error of the mean is shown. * - p > 0.001. ( E ) fkh2Δ cells expressing FKH1 K-R were arrested in S phase using HU, then released back into the cell cycle. Samples were removed every 30 minutes afterwards, treated +/− CHX for 1 hr, then proteins were recovered for Westerns. Bands from two experiments were scanned and the ratio of bands +/− CHX was plotted. See also Figure S3 .

    Journal: Aging (Albany NY)

    Article Title: Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance

    doi: 10.18632/aging.100949

    Figure Lengend Snippet: Mutation of the highly conserved K373 in Fkh1 stabilizes the protein in mitosis ( A ) Sequence alignment of Fkh1-like proteins across evolutionary boundaries highlighting the highly conserved lysine (K373). The mutation made is shown above in red. ( B ) Morphology of fkh2Δ cells harboring the FKH1 K-R mutation shown in ( A ). (C) fkh2Δ cells expressing FKH1 K-R were arrested in mitosis using nocodazole, then treated with CHX for 60 minutes. Proteins were then harvested and examined for Fkh1-TAP, Clb2 and GAPDH content. ( D ) All bands in ( C ) were imaged from three separate experiments and the ratio of intensities before and after CHX addition were determined. Standard error of the mean is shown. * - p > 0.001. ( E ) fkh2Δ cells expressing FKH1 K-R were arrested in S phase using HU, then released back into the cell cycle. Samples were removed every 30 minutes afterwards, treated +/− CHX for 1 hr, then proteins were recovered for Westerns. Bands from two experiments were scanned and the ratio of bands +/− CHX was plotted. See also Figure S3 .

    Article Snippet: For mitotic arrest, Nocodazole (Calbiochem) was used for approximately 3 hours, with 5 μg/ml added initially and then an additional 2.5 μg/ml added after 90 minutes.

    Techniques: Mutagenesis, Sequencing, Expressing

    CDK6 and CDC25A are downstream effectors of NANOG. (A) Quantitative RT-PCR analysis showing down-regulation of CDK6 and CDC25A in H1 NANOG, H9 NANOG, and hES-NCL1 NANOG sublines 42 h after the transfection of CDK6 and CDC25A siRNA. The data represent the mean ± SEM (error bars) from three experiments (one in each subline). The value for the control-transfected sample (vector only) was set to 1, and all other values were calculated with respect to this. (B) Western blotting showing down-regulation of CDK6 and CDC25A in H1 NANOG sublines. Molecular masses are indicated in kilodaltons. (C) Chart representation of the fraction of cells in S phase over time after transfection of CDK6 siRNA and synchronization by nocodazole for 18 h in the H9 NANOG subline assessed by propidium iodide staining. This figure represents an example of at least two independent experiments carried in the H9 subline. (D) Flow cytometry images showing retention of cells in G1 phase of the cell cycle after transfection of CDC25A and NANOG siRNAs and synchronization by nocodazole for 18 h in the H9 NANOG subline assessed by propidium iodide staining. This figure represents an example of at least two independent experiments performed in the H9 subline.

    Journal: The Journal of Cell Biology

    Article Title: A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and CDC25A

    doi: 10.1083/jcb.200801009

    Figure Lengend Snippet: CDK6 and CDC25A are downstream effectors of NANOG. (A) Quantitative RT-PCR analysis showing down-regulation of CDK6 and CDC25A in H1 NANOG, H9 NANOG, and hES-NCL1 NANOG sublines 42 h after the transfection of CDK6 and CDC25A siRNA. The data represent the mean ± SEM (error bars) from three experiments (one in each subline). The value for the control-transfected sample (vector only) was set to 1, and all other values were calculated with respect to this. (B) Western blotting showing down-regulation of CDK6 and CDC25A in H1 NANOG sublines. Molecular masses are indicated in kilodaltons. (C) Chart representation of the fraction of cells in S phase over time after transfection of CDK6 siRNA and synchronization by nocodazole for 18 h in the H9 NANOG subline assessed by propidium iodide staining. This figure represents an example of at least two independent experiments carried in the H9 subline. (D) Flow cytometry images showing retention of cells in G1 phase of the cell cycle after transfection of CDC25A and NANOG siRNAs and synchronization by nocodazole for 18 h in the H9 NANOG subline assessed by propidium iodide staining. This figure represents an example of at least two independent experiments performed in the H9 subline.

    Article Snippet: 24 h after transfection, hESCs were synchronized in G2 to M by incubation in 200 ng/ml of a nocodazole-containing medium for 18 h. The cells were washed three times with normal medium and collected by Accutase (Millipore) treatment at various time points as indicated in the results section.

    Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Staining, Flow Cytometry, Cytometry

    CDK6 and CDC25A regulate S phase in hESCs. (A) Quantitative RT-PCR analysis for the expression of CDK6 and CDC25A in H1, H9, and hES-NCL1 cell lines 42 h after the transfection of CDK6 and CDC25A siRNA. The data represent the mean ± SEM (error bars) from three independent experiments (one in each cell line). The value for the control siRNA was set to 1, and all other values were calculated with respect to this. (B) Down-regulation of CDK6 by flow cytometry 42 h after the transfection of CDK6 siRNAs in hESCs (a representative example from the H9 line is shown). (C) Down-regulation of CDC25A by Western blotting 42 h after the transfection of CDC25A siRNAs (a representative example from the H9 line is shown). Molecular masses are indicated in kilodaltons. (D) Reduction in CDK6 kinase activity upon knockdown of CDK6. The value for the control siRNA was set to 100%, and all other values were calculated with respect to this. (E) Reduction in CDC25A phosphatase activity upon knockdown of CDC25A. The value for the control siRNA was set to 100%, and all other values were calculated with respect to this. (D and E) The data represent the mean ± SEM from three experiments performed in the H9 cell line. (F) Flow cytometry images showing movement of cells through the cell cycle after transfection of CDK6 siRNAs and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (G) Chart representation of the fraction of cells in S phase over time after transfection of CDK6 siRNA and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (H) Flow cytometry images showing retention of cells in G1 phase of the cell cycle after transfection of CDC25A siRNA and NANOG siRNA and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (F–H) The figures represent an example of at least two independent experiments performed in the H9 subline.

    Journal: The Journal of Cell Biology

    Article Title: A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and CDC25A

    doi: 10.1083/jcb.200801009

    Figure Lengend Snippet: CDK6 and CDC25A regulate S phase in hESCs. (A) Quantitative RT-PCR analysis for the expression of CDK6 and CDC25A in H1, H9, and hES-NCL1 cell lines 42 h after the transfection of CDK6 and CDC25A siRNA. The data represent the mean ± SEM (error bars) from three independent experiments (one in each cell line). The value for the control siRNA was set to 1, and all other values were calculated with respect to this. (B) Down-regulation of CDK6 by flow cytometry 42 h after the transfection of CDK6 siRNAs in hESCs (a representative example from the H9 line is shown). (C) Down-regulation of CDC25A by Western blotting 42 h after the transfection of CDC25A siRNAs (a representative example from the H9 line is shown). Molecular masses are indicated in kilodaltons. (D) Reduction in CDK6 kinase activity upon knockdown of CDK6. The value for the control siRNA was set to 100%, and all other values were calculated with respect to this. (E) Reduction in CDC25A phosphatase activity upon knockdown of CDC25A. The value for the control siRNA was set to 100%, and all other values were calculated with respect to this. (D and E) The data represent the mean ± SEM from three experiments performed in the H9 cell line. (F) Flow cytometry images showing movement of cells through the cell cycle after transfection of CDK6 siRNAs and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (G) Chart representation of the fraction of cells in S phase over time after transfection of CDK6 siRNA and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (H) Flow cytometry images showing retention of cells in G1 phase of the cell cycle after transfection of CDC25A siRNA and NANOG siRNA and synchronization by nocodazole for 18 h assessed by propidium iodide staining. (F–H) The figures represent an example of at least two independent experiments performed in the H9 subline.

    Article Snippet: 24 h after transfection, hESCs were synchronized in G2 to M by incubation in 200 ng/ml of a nocodazole-containing medium for 18 h. The cells were washed three times with normal medium and collected by Accutase (Millipore) treatment at various time points as indicated in the results section.

    Techniques: Quantitative RT-PCR, Expressing, Transfection, Flow Cytometry, Cytometry, Western Blot, Activity Assay, Staining

    Changes in hESC cell cycle distribution as a result of NANOG overexpression. (A) Flow cytometry images showing movement of cells through the cell cycle after synchronization by nocodazole for 18 h assessed by propidium iodide staining. This figure represents an example of three independent experiments. (B) Chart representation of the fraction of cells in S phase assessed by propidium iodide staining over time after release from synchronization with nocodazole for 18 h. (C) Chart representation of the fraction of cells in S phase assessed by BrdU incorporation over time after release from synchronization with nocodazole for 18 h. The bottom panel shows flow cytometry images of BrdU-incorporating cells in NANOG and control H9 hESC sublines after 5 h of release from nocodazole. The dashed line represents the end of G1 and the start of S phase. (B and C) The figure represents an example of three independent experiments performed in H9 sublines. (D) Western blotting for expression of main components involved in G1 to S transition in NANOG-overexpressing and control clones. This summary is a representation of three independent experiments. GAPDH was used as a loading control. p16 and p15 were absent in all sublines, whereas p19 and p18 were expressed at very low but equal levels in NANOG-overexpressing and control clones (not depicted). Molecular masses are indicated in kilodaltons. HN, H1 NANOG; HC, H1 control; NN, hES-NCL1 NANOG; NC, hES-NCL1 control.

    Journal: The Journal of Cell Biology

    Article Title: A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and CDC25A

    doi: 10.1083/jcb.200801009

    Figure Lengend Snippet: Changes in hESC cell cycle distribution as a result of NANOG overexpression. (A) Flow cytometry images showing movement of cells through the cell cycle after synchronization by nocodazole for 18 h assessed by propidium iodide staining. This figure represents an example of three independent experiments. (B) Chart representation of the fraction of cells in S phase assessed by propidium iodide staining over time after release from synchronization with nocodazole for 18 h. (C) Chart representation of the fraction of cells in S phase assessed by BrdU incorporation over time after release from synchronization with nocodazole for 18 h. The bottom panel shows flow cytometry images of BrdU-incorporating cells in NANOG and control H9 hESC sublines after 5 h of release from nocodazole. The dashed line represents the end of G1 and the start of S phase. (B and C) The figure represents an example of three independent experiments performed in H9 sublines. (D) Western blotting for expression of main components involved in G1 to S transition in NANOG-overexpressing and control clones. This summary is a representation of three independent experiments. GAPDH was used as a loading control. p16 and p15 were absent in all sublines, whereas p19 and p18 were expressed at very low but equal levels in NANOG-overexpressing and control clones (not depicted). Molecular masses are indicated in kilodaltons. HN, H1 NANOG; HC, H1 control; NN, hES-NCL1 NANOG; NC, hES-NCL1 control.

    Article Snippet: 24 h after transfection, hESCs were synchronized in G2 to M by incubation in 200 ng/ml of a nocodazole-containing medium for 18 h. The cells were washed three times with normal medium and collected by Accutase (Millipore) treatment at various time points as indicated in the results section.

    Techniques: Over Expression, Flow Cytometry, Cytometry, Staining, BrdU Incorporation Assay, Western Blot, Expressing, Clone Assay

    Overexpression of CDK6 and CDC25A accelerates S-phase completion. (A) Quantitative RT-PCR for endogenous (left bars) and total (right bars) expression of CDK6 and CDC25A in the H9 hESC line. The data represent the mean ± SEM (error bars) from three independent experiments. The value for the control clone was set to 1, and all other values were calculated with respect to this. (B) Overexpression of CDC25A and CDC25A shown by Western blotting. GAPDH is used as a loading control (a representative example from the H9 line is shown). Molecular masses are indicated in kilodaltons. (C) Increase in CDK6 kinase activity and CDC25A phosphatase activity in CDK6- and CDC25A-overexpressing hESC sublines, respectively. The value for the control subline was set to 100%, and all other values were calculated with respect to this. The data represent the mean ± SEM from three experiments performed in the H9 cell line. (D) Chart representation of the fraction of cells in S phase over time in CDK6- and CDC25A-overexpressing hESC sublines assessed by propidium iodide staining (a representative example from the H9 subline is shown). (E) Flow cytometry images showing cell cycle distribution in H9 control as well as H9 CDK6 and H9 CDC25A after transfection of control siRNA and NANOG siRNA and synchronization by nocodazole for 18 h assessed by propidium iodide staining. This figure represents an example of at least two independent experiments performed in the H9 subline.

    Journal: The Journal of Cell Biology

    Article Title: A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and CDC25A

    doi: 10.1083/jcb.200801009

    Figure Lengend Snippet: Overexpression of CDK6 and CDC25A accelerates S-phase completion. (A) Quantitative RT-PCR for endogenous (left bars) and total (right bars) expression of CDK6 and CDC25A in the H9 hESC line. The data represent the mean ± SEM (error bars) from three independent experiments. The value for the control clone was set to 1, and all other values were calculated with respect to this. (B) Overexpression of CDC25A and CDC25A shown by Western blotting. GAPDH is used as a loading control (a representative example from the H9 line is shown). Molecular masses are indicated in kilodaltons. (C) Increase in CDK6 kinase activity and CDC25A phosphatase activity in CDK6- and CDC25A-overexpressing hESC sublines, respectively. The value for the control subline was set to 100%, and all other values were calculated with respect to this. The data represent the mean ± SEM from three experiments performed in the H9 cell line. (D) Chart representation of the fraction of cells in S phase over time in CDK6- and CDC25A-overexpressing hESC sublines assessed by propidium iodide staining (a representative example from the H9 subline is shown). (E) Flow cytometry images showing cell cycle distribution in H9 control as well as H9 CDK6 and H9 CDC25A after transfection of control siRNA and NANOG siRNA and synchronization by nocodazole for 18 h assessed by propidium iodide staining. This figure represents an example of at least two independent experiments performed in the H9 subline.

    Article Snippet: 24 h after transfection, hESCs were synchronized in G2 to M by incubation in 200 ng/ml of a nocodazole-containing medium for 18 h. The cells were washed three times with normal medium and collected by Accutase (Millipore) treatment at various time points as indicated in the results section.

    Techniques: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Activity Assay, Staining, Flow Cytometry, Cytometry, Transfection