nocodazole  (Millipore)


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    Name:
    Nocodazole
    Description:

    Catalog Number:
    m1404
    Price:
    None
    Applications:
    Nocodazole has been shown to enhance CRISPR genome editing efficiency. To see other small molecule CRISPR enhancers, visit sigma.com/CRISPR-enhancers.
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    Structured Review

    Millipore nocodazole
    Nocodazole

    https://www.bioz.com/result/nocodazole/product/Millipore
    Average 99 stars, based on 494 article reviews
    Price from $9.99 to $1999.99
    nocodazole - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Trackosome: a computational toolbox to study the spatiotemporal dynamics of centrosomes, nuclear envelope and cellular membrane"

    Article Title: Trackosome: a computational toolbox to study the spatiotemporal dynamics of centrosomes, nuclear envelope and cellular membrane

    Journal: bioRxiv

    doi: 10.1101/2020.04.27.064204

    Nuclear membrane fluctuations vary with the stage of the cell cycle and the physiological treatment. (A) Representative nucleus of each group. The phase of the cell cycle is evidenced by the marked histone (red), taken from the first frame of each video. The nuclear envelope (green) is shown at two different times stamps to illustrate the degree of membrane undulations in each group. Scale bar: 5 μm. (B) Median of the majorant frequency dependent fluctuations, u f , obtained for groups of cells in interphase and early mitosis. The curve for cells fixed with formaldehyde was also included to set the noise limit. (C) Median of the majorant u f obtained for groups of cells in interphase and mitosis, treated with DMSO, nocodazole (NOC) and fixed with formaldehyde. NOC caused a significant decrease of the membrane fluctuations in mitosis. (D) Median across cells of the average FT of the squared fluctuations of each cell,
    Figure Legend Snippet: Nuclear membrane fluctuations vary with the stage of the cell cycle and the physiological treatment. (A) Representative nucleus of each group. The phase of the cell cycle is evidenced by the marked histone (red), taken from the first frame of each video. The nuclear envelope (green) is shown at two different times stamps to illustrate the degree of membrane undulations in each group. Scale bar: 5 μm. (B) Median of the majorant frequency dependent fluctuations, u f , obtained for groups of cells in interphase and early mitosis. The curve for cells fixed with formaldehyde was also included to set the noise limit. (C) Median of the majorant u f obtained for groups of cells in interphase and mitosis, treated with DMSO, nocodazole (NOC) and fixed with formaldehyde. NOC caused a significant decrease of the membrane fluctuations in mitosis. (D) Median across cells of the average FT of the squared fluctuations of each cell,

    Techniques Used:

    2) Product Images from "BubR1 Is Modified by Sumoylation during Mitotic Progression"

    Article Title: BubR1 Is Modified by Sumoylation during Mitotic Progression

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.318261

    BubR1 is modified, resulting in a significant mobility shift on denaturing gels. A , HeLa cells were treated with nocodazole for (40 ng/ml) 14 h after which mitotic cells were collected by shake-off. These cells were then re-cultured in the presence or
    Figure Legend Snippet: BubR1 is modified, resulting in a significant mobility shift on denaturing gels. A , HeLa cells were treated with nocodazole for (40 ng/ml) 14 h after which mitotic cells were collected by shake-off. These cells were then re-cultured in the presence or

    Techniques Used: Modification, Mobility Shift, Cell Culture

    BubR1 sumoylation is associated with its degradation. A , HeLa cells treated with nocodazole for various times as indicated after which equal amounts of cell lysates were blotted for BubR1, PARP-1, and β-actin. p89 represents a cleaved fragment
    Figure Legend Snippet: BubR1 sumoylation is associated with its degradation. A , HeLa cells treated with nocodazole for various times as indicated after which equal amounts of cell lysates were blotted for BubR1, PARP-1, and β-actin. p89 represents a cleaved fragment

    Techniques Used:

    3) Product Images from "The microtubule-associated protein HURP recruits the centrosomal protein TACC3 to regulate K-fiber formation and support chromosome congression"

    Article Title: The microtubule-associated protein HURP recruits the centrosomal protein TACC3 to regulate K-fiber formation and support chromosome congression

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003676

    HURP modulates TACC3 to be involved in kinetochore MTs assembly. A , control and HURP-depleted HeLa cells were fixed 3, 5, and 10 min after release from a 5-h treatment with 1 μg/ml nocodazole and labeled with anti-HURP ( green ) and anti-α-tubulin ( red ) antibodies and DNA ( blue ). B , quantification of microtubule foci around the chromosome region in control cells ( n = 70 cells) and HURP-depleted cells ( n = 66 cells) fixed at 5 min. *, p
    Figure Legend Snippet: HURP modulates TACC3 to be involved in kinetochore MTs assembly. A , control and HURP-depleted HeLa cells were fixed 3, 5, and 10 min after release from a 5-h treatment with 1 μg/ml nocodazole and labeled with anti-HURP ( green ) and anti-α-tubulin ( red ) antibodies and DNA ( blue ). B , quantification of microtubule foci around the chromosome region in control cells ( n = 70 cells) and HURP-depleted cells ( n = 66 cells) fixed at 5 min. *, p

    Techniques Used: Labeling

    4) Product Images from "Spatial distribution of IL4 controls iNKT cell-DC crosstalk in tumors"

    Article Title: Spatial distribution of IL4 controls iNKT cell-DC crosstalk in tumors

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/s41423-019-0243-z

    Microtubule-organizing center (MTOC) controls polarized secretion of interleukin-4 (IL4). a Method to quantify the distance from the MTOC to the synaptic interface (SI). b , c Polarization of the MTOC in invariant natural killer T (iNKT) cells activated by distinct antigen variant-pulsed splenic dendritic cells (DCs) at the indicated time points. Scale bars, 2 μm. Data are representative of three independent experiments and more than 60 cells per group. d Distance from the MTOC to the SI in the iNKT cells described in b , c . Data are presented as the mean ± SEM of more than 40 cells per group. e Distance from the MTOC to the SI in iNKT cells activated by distinct antigen variant-pulsed RBL.CD1d cells at the indicated time points. Data are presented as the mean ± SEM of more than 60 cells per group. f , g Influences of nocodazole (33 μM) on IL4 secretory sites ( f ) and the frequency of iNKT cells with polarized secretion ( g ) after activation by α-galactosylceramide (αGC)-pulsed RBL.CD1d cells for 4 h. Scale bars, 2 μm. Data are representative of three independent experiments ( f ) or are presented as the mean ± SEM of three independent experiments ( g ). h – j Influences of taxol (100 nM) on the distance from the MTOC to the SI ( h , i ) and on IL4 polarization ( h , j ) in iNKT cells activated by αGC acC20:2-pulsed RBL.CD1d cells for 4 h. Scale bars, 2 μm. Data are representative of three independent experiments ( h ), are presented as the mean ± SEM of ( j ) three independent experiments or are presented as the mean ± SEM of more than 60 cells per group ( i ). Dotted lines indicate cell boundaries. Statistical analysis was performed using one-way analysis of variance (ANOVA) with the Tukey’s post test or using Student’s t test. * P
    Figure Legend Snippet: Microtubule-organizing center (MTOC) controls polarized secretion of interleukin-4 (IL4). a Method to quantify the distance from the MTOC to the synaptic interface (SI). b , c Polarization of the MTOC in invariant natural killer T (iNKT) cells activated by distinct antigen variant-pulsed splenic dendritic cells (DCs) at the indicated time points. Scale bars, 2 μm. Data are representative of three independent experiments and more than 60 cells per group. d Distance from the MTOC to the SI in the iNKT cells described in b , c . Data are presented as the mean ± SEM of more than 40 cells per group. e Distance from the MTOC to the SI in iNKT cells activated by distinct antigen variant-pulsed RBL.CD1d cells at the indicated time points. Data are presented as the mean ± SEM of more than 60 cells per group. f , g Influences of nocodazole (33 μM) on IL4 secretory sites ( f ) and the frequency of iNKT cells with polarized secretion ( g ) after activation by α-galactosylceramide (αGC)-pulsed RBL.CD1d cells for 4 h. Scale bars, 2 μm. Data are representative of three independent experiments ( f ) or are presented as the mean ± SEM of three independent experiments ( g ). h – j Influences of taxol (100 nM) on the distance from the MTOC to the SI ( h , i ) and on IL4 polarization ( h , j ) in iNKT cells activated by αGC acC20:2-pulsed RBL.CD1d cells for 4 h. Scale bars, 2 μm. Data are representative of three independent experiments ( h ), are presented as the mean ± SEM of ( j ) three independent experiments or are presented as the mean ± SEM of more than 60 cells per group ( i ). Dotted lines indicate cell boundaries. Statistical analysis was performed using one-way analysis of variance (ANOVA) with the Tukey’s post test or using Student’s t test. * P

    Techniques Used: Variant Assay, Activation Assay

    5) Product Images from "HPV18 E1^E4 is assembled into aggresome-like compartment and involved in sequestration of viral oncoproteins."

    Article Title: HPV18 E1^E4 is assembled into aggresome-like compartment and involved in sequestration of viral oncoproteins.

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2013.00251

    Dynein-dependent transport through microtubule filaments was required for the assembly of 18E1^E4-aggresome. (A) Nocodazole treatment disrupted the 18E1^E4-aggresome assembly. HeLa cells were transfected with FLAG-18E1^E4 expression plasmids, treated by nocodazole (10 mM) at 24 h after transfection. At 24 h after the treatment, the cells were fixed by 4% PFA, then stained with anti-FLAG antibody (red). Control shows untransfected cells, and “mock” indicates mock-treated cells. (B) γ-tubulin (green) was associated with small aggregates of FLAG-18E1^E4 (red) in the nocodazole-treated cells. The association could be detected in most of the cells (≥90%) that were positive for FLAG-18E1^E4 expression. Cells were prepared and treated as in (A) , except for fixation by cold methanol. (C , D) Ciliobrevin D (a dynein inhibitor) and tubacin (an HDAC6 inhibitor) treatments prevented 18E1^E4-aggresome assembly. Cells were prepared, fixed, and stained as in (A) , except for the inhibitors.
    Figure Legend Snippet: Dynein-dependent transport through microtubule filaments was required for the assembly of 18E1^E4-aggresome. (A) Nocodazole treatment disrupted the 18E1^E4-aggresome assembly. HeLa cells were transfected with FLAG-18E1^E4 expression plasmids, treated by nocodazole (10 mM) at 24 h after transfection. At 24 h after the treatment, the cells were fixed by 4% PFA, then stained with anti-FLAG antibody (red). Control shows untransfected cells, and “mock” indicates mock-treated cells. (B) γ-tubulin (green) was associated with small aggregates of FLAG-18E1^E4 (red) in the nocodazole-treated cells. The association could be detected in most of the cells (≥90%) that were positive for FLAG-18E1^E4 expression. Cells were prepared and treated as in (A) , except for fixation by cold methanol. (C , D) Ciliobrevin D (a dynein inhibitor) and tubacin (an HDAC6 inhibitor) treatments prevented 18E1^E4-aggresome assembly. Cells were prepared, fixed, and stained as in (A) , except for the inhibitors.

    Techniques Used: Transfection, Expressing, Staining

    Major viral oncoproteins were sequestrated in 18E1^E4-aggresome. (A) CV1 cells were co-transfected with 18E1^E4 and FLAG-tagged 18E5, 18E6, or 18E7 expression plasmids. At 48 h after transfection, cells were lysed by triple detergent buffer. The expression levels of HPV18 E5, E6, and E7 were analyzed both in soluble (left panel) and insoluble fractions (right panel) of cell lysates. (B) The effect of nocodazole treatment (10 mM) was examined by a similar experiment as shown in (A) . (C) Intracellular localization of FLAG-tagged 18E5, 18E6, or 18E7 (red) with EGFP-tagged 18E1^E4 (green) in CV1 cells. Nuclei were stained with DAPI. The colocalization could be detected in most of the cells (≥90%) that were positive for EGFP-tagged 18E1^E4 expression.
    Figure Legend Snippet: Major viral oncoproteins were sequestrated in 18E1^E4-aggresome. (A) CV1 cells were co-transfected with 18E1^E4 and FLAG-tagged 18E5, 18E6, or 18E7 expression plasmids. At 48 h after transfection, cells were lysed by triple detergent buffer. The expression levels of HPV18 E5, E6, and E7 were analyzed both in soluble (left panel) and insoluble fractions (right panel) of cell lysates. (B) The effect of nocodazole treatment (10 mM) was examined by a similar experiment as shown in (A) . (C) Intracellular localization of FLAG-tagged 18E5, 18E6, or 18E7 (red) with EGFP-tagged 18E1^E4 (green) in CV1 cells. Nuclei were stained with DAPI. The colocalization could be detected in most of the cells (≥90%) that were positive for EGFP-tagged 18E1^E4 expression.

    Techniques Used: Transfection, Expressing, Staining

    6) Product Images from "Quantitative Analysis of the Microtubule Interaction of Rabies Virus P3 Protein: Roles in Immune Evasion and Pathogenesis"

    Article Title: Quantitative Analysis of the Microtubule Interaction of Rabies Virus P3 Protein: Roles in Immune Evasion and Pathogenesis

    Journal: Scientific Reports

    doi: 10.1038/srep33493

    Interaction of Ni-P3 with MTs is impaired by N 226 -H mutation. ( a ) COS-7 cells were transfected to express the indicated proteins before analysis by live-cell CLSM; each image is representative of cells in 30 fields of view sampled over 3 separate assays (COS-7) or 9 fields of view (NSC-34). ( b–d ) COS-7 cells transfected to express GFP-Ni-P3 were treated with or without Taxol or nocodazole ( b ) co-transfected to express mCherry-tubulin ( c ) or fixed and immunostained for β-tubulin ( d ) before analysis by CLSM; colocalization in b and d is apparent as yellow coloration in merged image. ( e ) Live COS-7 cells expressing the indicated proteins were analyzed by CLSM to generate deconvoluted 3D images (images show reconstructed 3D images viewed down the z-axis). ( f ) Images such as those shown in ( e ) were analyzed to derive mean filament length values (mFL ± SEM; n ≥ 30 cells from 3 identical assays). p values were determined using the Mann Whitney test.
    Figure Legend Snippet: Interaction of Ni-P3 with MTs is impaired by N 226 -H mutation. ( a ) COS-7 cells were transfected to express the indicated proteins before analysis by live-cell CLSM; each image is representative of cells in 30 fields of view sampled over 3 separate assays (COS-7) or 9 fields of view (NSC-34). ( b–d ) COS-7 cells transfected to express GFP-Ni-P3 were treated with or without Taxol or nocodazole ( b ) co-transfected to express mCherry-tubulin ( c ) or fixed and immunostained for β-tubulin ( d ) before analysis by CLSM; colocalization in b and d is apparent as yellow coloration in merged image. ( e ) Live COS-7 cells expressing the indicated proteins were analyzed by CLSM to generate deconvoluted 3D images (images show reconstructed 3D images viewed down the z-axis). ( f ) Images such as those shown in ( e ) were analyzed to derive mean filament length values (mFL ± SEM; n ≥ 30 cells from 3 identical assays). p values were determined using the Mann Whitney test.

    Techniques Used: Mutagenesis, Transfection, Confocal Laser Scanning Microscopy, Expressing, MANN-WHITNEY

    7) Product Images from "Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1"

    Article Title: Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23329

    SAC maintenance and resolution in HeLa and hTERT-RPE1 cells treated with the phosphatase inhibitor Okadaic Acid Nocodazole-treated, prometaphase-arrested, ( A ) HeLa and ( B ) hTERT-RPE1 cells were collected. Upon nocodazole wash out cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus OA at 0.5 µM (Noco− OA 0.5), fresh medium plus OA at 1 µM (Noco− OA 1), fresh medium plus nocodazole and OA at 0.5 µM (Noco+ OA 0.5) and fresh medium plus nocodazole and OA at 1 µM (Noco+ OA 1). Nocodazole-treated, prometaphase-arrested ( C ) HeLa and ( D ) hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were divided into two sets, one received vehicle as control (Control) and the other OA at 0.5 µM (OA 0.5). Cells were then taken at the indicated time points of further incubation. Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). The data shown are representative of four independent experiments per type performed under identical conditions and giving similar results.
    Figure Legend Snippet: SAC maintenance and resolution in HeLa and hTERT-RPE1 cells treated with the phosphatase inhibitor Okadaic Acid Nocodazole-treated, prometaphase-arrested, ( A ) HeLa and ( B ) hTERT-RPE1 cells were collected. Upon nocodazole wash out cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus OA at 0.5 µM (Noco− OA 0.5), fresh medium plus OA at 1 µM (Noco− OA 1), fresh medium plus nocodazole and OA at 0.5 µM (Noco+ OA 0.5) and fresh medium plus nocodazole and OA at 1 µM (Noco+ OA 1). Nocodazole-treated, prometaphase-arrested ( C ) HeLa and ( D ) hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were divided into two sets, one received vehicle as control (Control) and the other OA at 0.5 µM (OA 0.5). Cells were then taken at the indicated time points of further incubation. Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). The data shown are representative of four independent experiments per type performed under identical conditions and giving similar results.

    Techniques Used: Incubation, SDS Page

    PP2A activity does not affect MCC formation and implementation of SAC-dependent mitotic arrest HeLa cells were treated for 18 hours with RO3306 (10 µM). Upon RO3306 wash out, cells were released into: fresh medium (Noco− OA− LB-100-), fresh medium plus nocodazole (Noco+ OA− LB-100-), fresh medium plus nocodazole and LB-100 (Noco+ OA− LB-100+) and fresh medium plus nocodazole and OA (Noco+ OA+ LB-100-). Cell samples were taken at the indicated time points of further incubation and ( A ) Cdc20 Ips (Cdc20 Ip) were resolved on SDS/PAGE and subsequently probed by immunoblotting for the indicated antigens (a total cell lysate, Tot; and mock Ip, Mk Ip, were also included as control in the SDS/PAGE; the asterisk marks immunoglobulin signal, *Ig). ( B ) Total lysates were also probed for the indicated antigens. The data shown are representative of four independent experiments performed under identical conditions and giving similar results.
    Figure Legend Snippet: PP2A activity does not affect MCC formation and implementation of SAC-dependent mitotic arrest HeLa cells were treated for 18 hours with RO3306 (10 µM). Upon RO3306 wash out, cells were released into: fresh medium (Noco− OA− LB-100-), fresh medium plus nocodazole (Noco+ OA− LB-100-), fresh medium plus nocodazole and LB-100 (Noco+ OA− LB-100+) and fresh medium plus nocodazole and OA (Noco+ OA+ LB-100-). Cell samples were taken at the indicated time points of further incubation and ( A ) Cdc20 Ips (Cdc20 Ip) were resolved on SDS/PAGE and subsequently probed by immunoblotting for the indicated antigens (a total cell lysate, Tot; and mock Ip, Mk Ip, were also included as control in the SDS/PAGE; the asterisk marks immunoglobulin signal, *Ig). ( B ) Total lysates were also probed for the indicated antigens. The data shown are representative of four independent experiments performed under identical conditions and giving similar results.

    Techniques Used: Activity Assay, Incubation, SDS Page

    Effects of the PP2A inhibitor LB-100 on SAC maintenance and resolution in HeLa and hTERT-RPE1 cells Nocodazole-treated, prometaphase-arrested, HeLa cells were collected. Upon nocodazole wash out, cells were divided into two sets, one received vehicle (DMSO) as control and the other LB-100 (10 µM). Cells were then taken at the indicated time points of further incubation. Two cell samples in the control set also received again nocodazole (Noco) and one of them LB-100 (LB-100) and, then, were taken at the indicated time points of further incubation. ( A ) Total samples were separated on SDS/PAGE and immunoblotted for phosphorylated Cdk1 substrates (Cdk1 p-subs), pT481-PRC1, PRC1, cyclin B1 (Cyc B1) and Cdk1. ( B ) Optical pT481-PRC1 (triangles) and cyclin B1 (squares) signal density (arbitrary units; normalized for total PRC1 and Cdk1 optical density values, respectively) were plotted as percent of time 0 samples from control (open symbols) and LB-100-treated (filled symbols) cells. ( C ) Nocodazole-treated, prometaphase-arrested, hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus LB-100 (Noco– LB-100+) or fresh medium plus nocodazole and LB-100 (Noco+ LB-100+). Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). ( D ) Nocodazole-treated, prometaphase-arrested, HeLa cells were collected. Upon nocodazole wash out, cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus 10 µM LB-100 (Noco– LB-100+). Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). The data shown are representative of four independent experiments performed under identical conditions and giving similar results.
    Figure Legend Snippet: Effects of the PP2A inhibitor LB-100 on SAC maintenance and resolution in HeLa and hTERT-RPE1 cells Nocodazole-treated, prometaphase-arrested, HeLa cells were collected. Upon nocodazole wash out, cells were divided into two sets, one received vehicle (DMSO) as control and the other LB-100 (10 µM). Cells were then taken at the indicated time points of further incubation. Two cell samples in the control set also received again nocodazole (Noco) and one of them LB-100 (LB-100) and, then, were taken at the indicated time points of further incubation. ( A ) Total samples were separated on SDS/PAGE and immunoblotted for phosphorylated Cdk1 substrates (Cdk1 p-subs), pT481-PRC1, PRC1, cyclin B1 (Cyc B1) and Cdk1. ( B ) Optical pT481-PRC1 (triangles) and cyclin B1 (squares) signal density (arbitrary units; normalized for total PRC1 and Cdk1 optical density values, respectively) were plotted as percent of time 0 samples from control (open symbols) and LB-100-treated (filled symbols) cells. ( C ) Nocodazole-treated, prometaphase-arrested, hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus LB-100 (Noco– LB-100+) or fresh medium plus nocodazole and LB-100 (Noco+ LB-100+). Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). ( D ) Nocodazole-treated, prometaphase-arrested, HeLa cells were collected. Upon nocodazole wash out, cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus 10 µM LB-100 (Noco– LB-100+). Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). The data shown are representative of four independent experiments performed under identical conditions and giving similar results.

    Techniques Used: Incubation, SDS Page

    PP2A activity does not substantially affect progression beyond anaphase Nocodazole-treated, prometaphase-arrested, ( A ) HeLa and ( B ) hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were divided into three sets that received either vehicle as control (CTRL), OA at 0.5 µM (OA), or LB-100. Cells were then taken at the indicated time points of further incubation and spun onto microscopy slides and processed for immunofluorescence staining for α-tubulin and the centromere marker CREST, DNA was stained with Hoechst. Upper graphs: post-anaphase cells were visually scored through microscopy. Error bars refer to variation within three independent experiments performed under identical conditions. Lower photographs: indicative images of HeLa and hTERT-RPE1 cells taken at 60 or 40 min, respectively, of incubation. Scale bars, 10 μm.
    Figure Legend Snippet: PP2A activity does not substantially affect progression beyond anaphase Nocodazole-treated, prometaphase-arrested, ( A ) HeLa and ( B ) hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were divided into three sets that received either vehicle as control (CTRL), OA at 0.5 µM (OA), or LB-100. Cells were then taken at the indicated time points of further incubation and spun onto microscopy slides and processed for immunofluorescence staining for α-tubulin and the centromere marker CREST, DNA was stained with Hoechst. Upper graphs: post-anaphase cells were visually scored through microscopy. Error bars refer to variation within three independent experiments performed under identical conditions. Lower photographs: indicative images of HeLa and hTERT-RPE1 cells taken at 60 or 40 min, respectively, of incubation. Scale bars, 10 μm.

    Techniques Used: Activity Assay, Incubation, Microscopy, Immunofluorescence, Staining, Marker

    PP2A activity does not affect MCC maintenance and disassembly Nocodazole-treated, prometaphase-arrested, HeLa cells were washed out of nocodazole and further incubated for 60 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus LB-100 (10 µM; Noco− LB-100+), fresh medium plus OA (0.5 µM; Noco− OA+), fresh medium plus nocodazole and LB-100 (10 µM; Noco+ LB-100+) and fresh medium plus nocodazole and OA (0.5 µM; Noco+ OA+). Cdc20 was immunoprecipitated (Ip) from cell lysates. ( A ) Cdc20 Ips (Cdc20 Ip) were resolved on SDS/PAGE and subsequently probed by immunoblotting for the indicated antigens (a total cell lysate, Tot; and mock Ip, Mk Ip, were also included as control in the SDS/PAGE; the asterisk marks immunoglobulin signal, *Ig). ( B ) Total lysates were also probed for the indicated antigens. ( C ) Nocodazole-treated, prometaphase-arrested, HeLa cells were washed out of nocodazole and further incubated for 60 min in: fresh medium (LB-100- OA−), fresh medium plus LB-100 (10 µM; LB-100+ OA−) or fresh medium plus OA (0.5 µM; LB-100- OA+). BubR1 was probed on blots of long, higher resolving, SDS/PAGE runs of total lysates (short and long blot exposures are shown). Total lysates were also probed for the other indicated antigens. The data shown are representative of three independent experiments performed under identical conditions and giving similar results.
    Figure Legend Snippet: PP2A activity does not affect MCC maintenance and disassembly Nocodazole-treated, prometaphase-arrested, HeLa cells were washed out of nocodazole and further incubated for 60 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus LB-100 (10 µM; Noco− LB-100+), fresh medium plus OA (0.5 µM; Noco− OA+), fresh medium plus nocodazole and LB-100 (10 µM; Noco+ LB-100+) and fresh medium plus nocodazole and OA (0.5 µM; Noco+ OA+). Cdc20 was immunoprecipitated (Ip) from cell lysates. ( A ) Cdc20 Ips (Cdc20 Ip) were resolved on SDS/PAGE and subsequently probed by immunoblotting for the indicated antigens (a total cell lysate, Tot; and mock Ip, Mk Ip, were also included as control in the SDS/PAGE; the asterisk marks immunoglobulin signal, *Ig). ( B ) Total lysates were also probed for the indicated antigens. ( C ) Nocodazole-treated, prometaphase-arrested, HeLa cells were washed out of nocodazole and further incubated for 60 min in: fresh medium (LB-100- OA−), fresh medium plus LB-100 (10 µM; LB-100+ OA−) or fresh medium plus OA (0.5 µM; LB-100- OA+). BubR1 was probed on blots of long, higher resolving, SDS/PAGE runs of total lysates (short and long blot exposures are shown). Total lysates were also probed for the other indicated antigens. The data shown are representative of three independent experiments performed under identical conditions and giving similar results.

    Techniques Used: Activity Assay, Incubation, Immunoprecipitation, SDS Page

    8) Product Images from "ATP depletion during mitotic arrest induces mitotic slippage and APC/CCdh1-dependent cyclin B1 degradation"

    Article Title: ATP depletion during mitotic arrest induces mitotic slippage and APC/CCdh1-dependent cyclin B1 degradation

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-018-0069-2

    Mitotic slippage via APC/C-dependent degradation of cyclin B. a Mitotic arrested cell lysates from the indicated times after treatment with/without 2-DG and NaN 3 were subjected to western blot analysis using antibodies for cyclin B, securin, and actin (loading control). b Cell arrested in mitosis were transfected with GFP-cyclin B and treated with/without 2-DG and NaN 3 in the presence of nocodazole. Time-lapse images of cyclin B degradation are representative of 36 control cells and 47 cells treated with 2-DG and NaN 3 . Time is presented as minutes after cell rounding up for mitosis. The relative intensity of the GFP-cyclin B signal at different times was measured using NIS-Elements Viewer 4.0 software. The results are shown as the mean (control; black bar, 2-DG and NaN 3 ; white bar). c Cells arrested in mitosis were treated with/without 2-DG and NaN 3 (2 + N) in the absence/presence of MG132. Quantification of the percentage of mitotic cells with condensed chromosomes was performed by aceto-orcein staining. The results are shown as the mean ± SD from two independent experiments ( n = 100). d Cell lysates from the indicated times after treatment with/without 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies against cyclin B and actin (loading control). e For APC inhibition, cells arrested in mitosis were treated with 12 μM proTAME for 30 min prior to co-treatment with/without 2-DG and NaN 3 (2 + N). Quantification of the percentage of mitotic cells with condensed chromosome was performed by aceto-orcein staining. The results are shown as the mean ± SD from two independent experiments ( n = 100). f Cell lysates from the indicated times after treatment with 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies for cyclin B and GAPDH (loading control)
    Figure Legend Snippet: Mitotic slippage via APC/C-dependent degradation of cyclin B. a Mitotic arrested cell lysates from the indicated times after treatment with/without 2-DG and NaN 3 were subjected to western blot analysis using antibodies for cyclin B, securin, and actin (loading control). b Cell arrested in mitosis were transfected with GFP-cyclin B and treated with/without 2-DG and NaN 3 in the presence of nocodazole. Time-lapse images of cyclin B degradation are representative of 36 control cells and 47 cells treated with 2-DG and NaN 3 . Time is presented as minutes after cell rounding up for mitosis. The relative intensity of the GFP-cyclin B signal at different times was measured using NIS-Elements Viewer 4.0 software. The results are shown as the mean (control; black bar, 2-DG and NaN 3 ; white bar). c Cells arrested in mitosis were treated with/without 2-DG and NaN 3 (2 + N) in the absence/presence of MG132. Quantification of the percentage of mitotic cells with condensed chromosomes was performed by aceto-orcein staining. The results are shown as the mean ± SD from two independent experiments ( n = 100). d Cell lysates from the indicated times after treatment with/without 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies against cyclin B and actin (loading control). e For APC inhibition, cells arrested in mitosis were treated with 12 μM proTAME for 30 min prior to co-treatment with/without 2-DG and NaN 3 (2 + N). Quantification of the percentage of mitotic cells with condensed chromosome was performed by aceto-orcein staining. The results are shown as the mean ± SD from two independent experiments ( n = 100). f Cell lysates from the indicated times after treatment with 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies for cyclin B and GAPDH (loading control)

    Techniques Used: Western Blot, Transfection, Software, Staining, Inhibition

    APC/Cdh1, but not APC/Cdc20, is responsible for cyclin B degradation following ATP depletion. a After 20 h of Cdh1 siRNA transfection, nocodazole treatment was applied for 16 h. Mitotic cells obtained by shake off were treated with/without 2-DG and NaN 3 (2 + N). Quantification of the percentage of mitotic cells with condensed chromosome was performed by aceto-orcein staining. The results are given as the mean ± SD from three independent experiments ( n = 100). b The procedure was the same as in a . Cell lysates from the indicated times after treatment with 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies for cyclin B, Cdh1 and GAPDH. c Asynchronous cells ( a ) and mitotic arrested cell lysates from the indicated times after treatment with 2-DG and NaN 3 (2 + N) were subjected to immunoprecipitation using an antibody for Cdc27 and western blot analysis using antibodies for Cdc27, Cdh1, Cdc20, cyclin B, and GAPDH. d Quantification of the relative association with APC/C was performed by measuring the band intensity using ImageJ software after treatment with 2-DG and NaN 3 (2 + N). The results are given as the mean from five independent experiments
    Figure Legend Snippet: APC/Cdh1, but not APC/Cdc20, is responsible for cyclin B degradation following ATP depletion. a After 20 h of Cdh1 siRNA transfection, nocodazole treatment was applied for 16 h. Mitotic cells obtained by shake off were treated with/without 2-DG and NaN 3 (2 + N). Quantification of the percentage of mitotic cells with condensed chromosome was performed by aceto-orcein staining. The results are given as the mean ± SD from three independent experiments ( n = 100). b The procedure was the same as in a . Cell lysates from the indicated times after treatment with 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies for cyclin B, Cdh1 and GAPDH. c Asynchronous cells ( a ) and mitotic arrested cell lysates from the indicated times after treatment with 2-DG and NaN 3 (2 + N) were subjected to immunoprecipitation using an antibody for Cdc27 and western blot analysis using antibodies for Cdc27, Cdh1, Cdc20, cyclin B, and GAPDH. d Quantification of the relative association with APC/C was performed by measuring the band intensity using ImageJ software after treatment with 2-DG and NaN 3 (2 + N). The results are given as the mean from five independent experiments

    Techniques Used: Transfection, Staining, Western Blot, Immunoprecipitation, Software

    Decrease in Cdc20 before cyclin B in mitotic slippage following ATP depletion. a Comparison of the protein levels during mitotic slippage induced by 5 nM hesperadin (Hes), 1 μM RO3306 (RO), or co-treatment with 2-DG and NaN 3 (2 + N). Cells arrested in mitosis by nocodazole treatment for 16 h were treated with each drug for the indicated times. Cell lysates from the indicated times after drug treatment were subjected to western blot analysis using antibodies for cyclin B, Cdc20, and GAPDH (loading control). b Cells arrested in mitosis for 16 h were released. Cell lysates from the indicated times after release from nocodazole were subjected to western blot analysis using antibodies for cyclin B, Cdc20 and α-tubulin (loading control). c Mitotic arrested cell lysates from the indicated times after treatment with/without 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies for cyclin B, Cdc20, phospho-Histone H3Ser10, Aurora B, Plk1, Cdh1, phospho-p70S6 Kinase, and GAPDH (loading control). *non-specific band
    Figure Legend Snippet: Decrease in Cdc20 before cyclin B in mitotic slippage following ATP depletion. a Comparison of the protein levels during mitotic slippage induced by 5 nM hesperadin (Hes), 1 μM RO3306 (RO), or co-treatment with 2-DG and NaN 3 (2 + N). Cells arrested in mitosis by nocodazole treatment for 16 h were treated with each drug for the indicated times. Cell lysates from the indicated times after drug treatment were subjected to western blot analysis using antibodies for cyclin B, Cdc20, and GAPDH (loading control). b Cells arrested in mitosis for 16 h were released. Cell lysates from the indicated times after release from nocodazole were subjected to western blot analysis using antibodies for cyclin B, Cdc20 and α-tubulin (loading control). c Mitotic arrested cell lysates from the indicated times after treatment with/without 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies for cyclin B, Cdc20, phospho-Histone H3Ser10, Aurora B, Plk1, Cdh1, phospho-p70S6 Kinase, and GAPDH (loading control). *non-specific band

    Techniques Used: Western Blot

    Induction of mitotic slippage by ATP depletion. Cells incubated with 100 ng/ml nocodazole for 16 h were treated with/without 6 mM 2-deoxyglucose (2-DG) and 10 mM sodium azide (NaN 3 ). a The relative ATP levels of cells were measured at the indicated time points after 2-DG and NaN 3 treatment. The results are given as the mean ± SD from three independent experiments. *** P
    Figure Legend Snippet: Induction of mitotic slippage by ATP depletion. Cells incubated with 100 ng/ml nocodazole for 16 h were treated with/without 6 mM 2-deoxyglucose (2-DG) and 10 mM sodium azide (NaN 3 ). a The relative ATP levels of cells were measured at the indicated time points after 2-DG and NaN 3 treatment. The results are given as the mean ± SD from three independent experiments. *** P

    Techniques Used: Incubation

    Decreased protein synthesis by ATP depletion and the short half-life reduced the level of cdc20 during mitotic slippage. a Mitotic arrested cells were treated with 2-DG and NaN 3 (2 + N), and expression of Cdc20 mRNA was analyzed by real-time PCR. The results are given as the mean ± SD from three independent experiments. b Experimental design (upper panel). For protein synthesis experiments, HeLa cells grown in DMEM/high glucose were used. Cells arrested in mitosis were obtained by nocodazole treatment (100 ng/ml) for 15 h followed by shake-off, and then were incubated in methionine-free medium with nocodazole. After 1 h, cells were pre-exposed to 2-DG and NaN 3 (2 + N) for 30 min. [ 35 S]-methionine was added to the medium, and proteins were labeled for 2 h and subsequently chased by the addition of an excess of unlabeled methionine for 1 h, 2 h and 4 h. Total cell lysates and immunoprecipitated lysates using Cdc20 or Cdh1 antibody were resolved by SDS-PAGE and visualized by Coomassie Blue staining or autoradiography. c Mitotic arrested cell lysates from the indicated times after co-treatment with 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies for Cdc20, phospho-p70S6 kinase, and GAPDH. d Quantification of the relative intensity of Cdc20 or Cdh1 was performed by measuring the band intensity using ImageJ software. e For inhibition of protein synthesis, cells were treated with 10 μg/ml cycloheximide for 30 min prior to co-treatment with 2-DG and NaN 3 . Cell lysates from the indicated times after co-treatment with 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies for Cdc20, Cdh1, and GAPDH (upper panel). Quantification of the relative intensity of Cdc20 or Cdh1 was performed by measuring the band intensity using ImageJ software (lower panel)
    Figure Legend Snippet: Decreased protein synthesis by ATP depletion and the short half-life reduced the level of cdc20 during mitotic slippage. a Mitotic arrested cells were treated with 2-DG and NaN 3 (2 + N), and expression of Cdc20 mRNA was analyzed by real-time PCR. The results are given as the mean ± SD from three independent experiments. b Experimental design (upper panel). For protein synthesis experiments, HeLa cells grown in DMEM/high glucose were used. Cells arrested in mitosis were obtained by nocodazole treatment (100 ng/ml) for 15 h followed by shake-off, and then were incubated in methionine-free medium with nocodazole. After 1 h, cells were pre-exposed to 2-DG and NaN 3 (2 + N) for 30 min. [ 35 S]-methionine was added to the medium, and proteins were labeled for 2 h and subsequently chased by the addition of an excess of unlabeled methionine for 1 h, 2 h and 4 h. Total cell lysates and immunoprecipitated lysates using Cdc20 or Cdh1 antibody were resolved by SDS-PAGE and visualized by Coomassie Blue staining or autoradiography. c Mitotic arrested cell lysates from the indicated times after co-treatment with 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies for Cdc20, phospho-p70S6 kinase, and GAPDH. d Quantification of the relative intensity of Cdc20 or Cdh1 was performed by measuring the band intensity using ImageJ software. e For inhibition of protein synthesis, cells were treated with 10 μg/ml cycloheximide for 30 min prior to co-treatment with 2-DG and NaN 3 . Cell lysates from the indicated times after co-treatment with 2-DG and NaN 3 (2 + N) were subjected to western blot analysis using antibodies for Cdc20, Cdh1, and GAPDH (upper panel). Quantification of the relative intensity of Cdc20 or Cdh1 was performed by measuring the band intensity using ImageJ software (lower panel)

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation, Labeling, Immunoprecipitation, SDS Page, Staining, Autoradiography, Western Blot, Software, Inhibition

    9) Product Images from "Self-Assembly of the RZZ Complex into Filaments Drives Kinetochore Expansion in the Absence of Microtubule Attachment"

    Article Title: Self-Assembly of the RZZ Complex into Filaments Drives Kinetochore Expansion in the Absence of Microtubule Attachment

    Journal: Current Biology

    doi: 10.1016/j.cub.2018.08.056

    C. elegans ROD-1 Is Capable of Self-Assembly into Micrometer-Scale Filaments In Vivo (A) (Left) Schematic of experimental protocol to visualize kinetochore expansion in C. elegans early embryos. RNAi-mediated depletion of PERM-1 permeabilizes the eggshell of embryos, which are subsequently isolated from hermaphrodite adults, treated with nocodazole, and immunostained for ROD-1 and the centromere marker GFP::HCP-3 CENP-A . (Right) Immunofluorescence images of mitotic embryonic cells with and without nocodazole treatment. Arrows point at filamentous kinetochore expansions containing ROD-1 that form in the absence of microtubules. Scale bars, 2 μm. (B) (Left) Schematic of the C. elegans early embryo at the two-, four-, and eight-cell stages. Names of individual cells are indicated. Bars connecting cells indicate they originated from the same mother cell. (Right) Selected images from a time-lapse sequence of an early embryo expressing endogenous ROD-1 tagged with GFP. GFP::ROD-1 is enriched in nuclei and localizes transiently to holocentric kinetochores in mitosis. In addition, GFP::ROD-1 starts to form filaments during mitosis at the eight-cell stage, but not earlier (see also Video S1 ). Dashed lines mark cell boundaries. Scale bars, 5 μm; blow-up, 2 μm. (C) Selected images from a time-lapse sequence documenting the formation of GFP::ROD-1 filaments during mitosis at the eight-cell stage (see also Video S2 ). mCherry::histone H2B labels chromosomes. Filaments, typically several micrometers in length, form in the nucleus before NEBD and segregate to daughter cells by clustering at spindle poles. Kinetochore-localized GFP::ROD-1 is also visible (arrows). Time point 0 refers to the last frame before the appearance of GFP::ROD-1 on filaments and kinetochores. Scale bar, 2 μm. (D) (Left) Schematic highlighting the increase in nuclear GFP::ROD-1 levels during early embryonic development. (Right) Selected images of nuclei from a time-lapse sequence of a developing embryo expressing GFP::ROD-1 that was followed from the two-cell stage to the eight-cell stage. (Top) Images show the EMS cell in the four-cell embryo, which gives rise to the MS cell in the eight-cell embryo. (Bottom) Likewise, the ABp cell gives rise to the ABpl cell. In both instances, nuclear GFP::ROD-1 levels increase gradually during the cell cycle and are significantly higher in daughter cells. Time point 0 denotes the last frame before GFP::ROD-1 appears on kinetochores (EMS and ABp) or filaments (MS and ABpl). Similar results were obtained for nuclei of the P lineage (not shown). Scale bar, 2 μm. (E) Quantification of average GFP::ROD-1 signal in nuclei and the cytoplasm in developing embryos as shown in (D). Average fluorescence intensity was determined in images acquired every 20 s, averaged for the indicated number n of cells from at least 8 embryos, and plotted against time. Time point 0 denotes the last frame before the appearance of GFP::ROD-1 on filaments and/or kinetochores. Values are shown as mean ± 95% confidence interval for nuclear signal and as the mean for cytoplasmic signal. (F) (Left) Quantification of nuclear GFP::ROD-1 levels in cells at different developmental stages. Measurements correspond to the last frame before GFP::ROD-1 appears on filaments and/or kinetochores (time point 0 s), showing a significant increase of nuclear signal at the eight-cell stage. Mild rod-1(RNAi) was used to reduce GFP::ROD-1 levels. Values are shown as mean ± 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ∗∗∗∗ p
    Figure Legend Snippet: C. elegans ROD-1 Is Capable of Self-Assembly into Micrometer-Scale Filaments In Vivo (A) (Left) Schematic of experimental protocol to visualize kinetochore expansion in C. elegans early embryos. RNAi-mediated depletion of PERM-1 permeabilizes the eggshell of embryos, which are subsequently isolated from hermaphrodite adults, treated with nocodazole, and immunostained for ROD-1 and the centromere marker GFP::HCP-3 CENP-A . (Right) Immunofluorescence images of mitotic embryonic cells with and without nocodazole treatment. Arrows point at filamentous kinetochore expansions containing ROD-1 that form in the absence of microtubules. Scale bars, 2 μm. (B) (Left) Schematic of the C. elegans early embryo at the two-, four-, and eight-cell stages. Names of individual cells are indicated. Bars connecting cells indicate they originated from the same mother cell. (Right) Selected images from a time-lapse sequence of an early embryo expressing endogenous ROD-1 tagged with GFP. GFP::ROD-1 is enriched in nuclei and localizes transiently to holocentric kinetochores in mitosis. In addition, GFP::ROD-1 starts to form filaments during mitosis at the eight-cell stage, but not earlier (see also Video S1 ). Dashed lines mark cell boundaries. Scale bars, 5 μm; blow-up, 2 μm. (C) Selected images from a time-lapse sequence documenting the formation of GFP::ROD-1 filaments during mitosis at the eight-cell stage (see also Video S2 ). mCherry::histone H2B labels chromosomes. Filaments, typically several micrometers in length, form in the nucleus before NEBD and segregate to daughter cells by clustering at spindle poles. Kinetochore-localized GFP::ROD-1 is also visible (arrows). Time point 0 refers to the last frame before the appearance of GFP::ROD-1 on filaments and kinetochores. Scale bar, 2 μm. (D) (Left) Schematic highlighting the increase in nuclear GFP::ROD-1 levels during early embryonic development. (Right) Selected images of nuclei from a time-lapse sequence of a developing embryo expressing GFP::ROD-1 that was followed from the two-cell stage to the eight-cell stage. (Top) Images show the EMS cell in the four-cell embryo, which gives rise to the MS cell in the eight-cell embryo. (Bottom) Likewise, the ABp cell gives rise to the ABpl cell. In both instances, nuclear GFP::ROD-1 levels increase gradually during the cell cycle and are significantly higher in daughter cells. Time point 0 denotes the last frame before GFP::ROD-1 appears on kinetochores (EMS and ABp) or filaments (MS and ABpl). Similar results were obtained for nuclei of the P lineage (not shown). Scale bar, 2 μm. (E) Quantification of average GFP::ROD-1 signal in nuclei and the cytoplasm in developing embryos as shown in (D). Average fluorescence intensity was determined in images acquired every 20 s, averaged for the indicated number n of cells from at least 8 embryos, and plotted against time. Time point 0 denotes the last frame before the appearance of GFP::ROD-1 on filaments and/or kinetochores. Values are shown as mean ± 95% confidence interval for nuclear signal and as the mean for cytoplasmic signal. (F) (Left) Quantification of nuclear GFP::ROD-1 levels in cells at different developmental stages. Measurements correspond to the last frame before GFP::ROD-1 appears on filaments and/or kinetochores (time point 0 s), showing a significant increase of nuclear signal at the eight-cell stage. Mild rod-1(RNAi) was used to reduce GFP::ROD-1 levels. Values are shown as mean ± 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ∗∗∗∗ p

    Techniques Used: In Vivo, Isolation, Marker, Immunofluorescence, Sequencing, Expressing, Mass Spectrometry, Fluorescence

    Kinetochore Expansion Requires the RZZ Complex and SPDL1 but Is Independent of Dynein-Dynactin (A) Cartoon showing the crescent shape characteristic of the expanded outer kinetochore, which encircles the compact inner kinetochore. Components analyzed in this figure are listed on the right. (B) Immunofluorescence images showing that kinetochore expansion in nocodazole is inhibited after RNAi-mediated depletion of ROD or SPDL1. Scale bars, 5 μm; blow-ups, 1 μm. (C–F) Quantification of relative kinetochore volume for CENP-E (C) and CENP-F (E) and kinetochore signal intensity for CENP-E (D) and CENP-F (F), based on fluorescence measurements in images as shown in (B). For each condition, the mean value per kinetochore was determined for individual cells. Final values are shown as the mean of mean (n = 20 cells), normalized to the control. Error bars denote the 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. ∗∗∗∗ p
    Figure Legend Snippet: Kinetochore Expansion Requires the RZZ Complex and SPDL1 but Is Independent of Dynein-Dynactin (A) Cartoon showing the crescent shape characteristic of the expanded outer kinetochore, which encircles the compact inner kinetochore. Components analyzed in this figure are listed on the right. (B) Immunofluorescence images showing that kinetochore expansion in nocodazole is inhibited after RNAi-mediated depletion of ROD or SPDL1. Scale bars, 5 μm; blow-ups, 1 μm. (C–F) Quantification of relative kinetochore volume for CENP-E (C) and CENP-F (E) and kinetochore signal intensity for CENP-E (D) and CENP-F (F), based on fluorescence measurements in images as shown in (B). For each condition, the mean value per kinetochore was determined for individual cells. Final values are shown as the mean of mean (n = 20 cells), normalized to the control. Error bars denote the 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. ∗∗∗∗ p

    Techniques Used: Immunofluorescence, Fluorescence

    10) Product Images from "microRNA 193a-5p Regulates Levels of Nucleolar- and Spindleassociated Protein 1 to Suppress Hepatocarcinogenesis"

    Article Title: microRNA 193a-5p Regulates Levels of Nucleolar- and Spindleassociated Protein 1 to Suppress Hepatocarcinogenesis

    Journal: Gastroenterology

    doi: 10.1053/j.gastro.2018.08.032

    NUSAP1 knockdown suppresses the cell growth and increases apoptotic cell death in Huh7 cells. ( A ) qRT-PCR and Western blot analyses of NUSAP1 in Huh7 cells transfected with siRNA against NUSAP1 or Control siRNA (n = 3 per group). ( B ) Quantification of colonies from Huh7 cells transfected with NUSAP1 or Control siRNA for 72 h (n = 3 per group) (bar = 1 cm). ( C ) Levels of cell-cycle proteins (Cyclin D1, Cyclin E1, Cyclin B1, Cyclin A2, p21) were detected in cell lysates of NUSAP1 siRNA transfected Huh7 cells by Western blot with GAPDH as a loading control (n = 3 per group). ( D ) Huh7 cells were transfected with siNUSAP1 for 72 h and analyzed with flow cytometry by DAPI staining (n = 6 per group). The percentage of cells in the Sub-G1, G 1 , S and G2/M phases of the cell cycle are indicated. ( E ) Huh7 cells were transfected with siNUSAP1 and treated with nocodazole (100 ng/ml) for 24 h before addition of DAPI and analyzed by FACS (n = 6 per group). The percentage of cells in the G 1 , S and G2/M phases of the cell cycle are indicated. ( F and G ) Representative images and quantification of TUNEL ( F ) and cl. Casp-3 ( G ) stainings in Huh7 cells transfected with NUSAP1 siRNA for 72 h (n = 3 per group) (bar = 50 μm). ( H ) Transwell migration assay for Huh7 cells was determined after transfection with NUSAP1 siRNA or Control (n = 3 per group) (bar = 50 μm). Results are represented as mean ± SEM. ns non-significant, * p
    Figure Legend Snippet: NUSAP1 knockdown suppresses the cell growth and increases apoptotic cell death in Huh7 cells. ( A ) qRT-PCR and Western blot analyses of NUSAP1 in Huh7 cells transfected with siRNA against NUSAP1 or Control siRNA (n = 3 per group). ( B ) Quantification of colonies from Huh7 cells transfected with NUSAP1 or Control siRNA for 72 h (n = 3 per group) (bar = 1 cm). ( C ) Levels of cell-cycle proteins (Cyclin D1, Cyclin E1, Cyclin B1, Cyclin A2, p21) were detected in cell lysates of NUSAP1 siRNA transfected Huh7 cells by Western blot with GAPDH as a loading control (n = 3 per group). ( D ) Huh7 cells were transfected with siNUSAP1 for 72 h and analyzed with flow cytometry by DAPI staining (n = 6 per group). The percentage of cells in the Sub-G1, G 1 , S and G2/M phases of the cell cycle are indicated. ( E ) Huh7 cells were transfected with siNUSAP1 and treated with nocodazole (100 ng/ml) for 24 h before addition of DAPI and analyzed by FACS (n = 6 per group). The percentage of cells in the G 1 , S and G2/M phases of the cell cycle are indicated. ( F and G ) Representative images and quantification of TUNEL ( F ) and cl. Casp-3 ( G ) stainings in Huh7 cells transfected with NUSAP1 siRNA for 72 h (n = 3 per group) (bar = 50 μm). ( H ) Transwell migration assay for Huh7 cells was determined after transfection with NUSAP1 siRNA or Control (n = 3 per group) (bar = 50 μm). Results are represented as mean ± SEM. ns non-significant, * p

    Techniques Used: Quantitative RT-PCR, Western Blot, Transfection, Flow Cytometry, Cytometry, Staining, FACS, TUNEL Assay, Transwell Migration Assay

    Overexpression of miR-193a-5p suppresses the progression of cell cycle in vitro . ( A ) Huh7 cells were transfected with miR-193a-5p mimic and Control siRNA and cell viability was analyzed by CCK-8 assay (n = 4 per group). ( B ) Representative immunofluorescence images and quantification of Ki67 staining of Huh7 cells transfected with miR-193a-5p mimic or Control siRNA (n = 3 per group) (bar = 50 μm). ( C ) Colony formation assay was performed after transfection of Huh7 cells with miR-193a-5p mimic or Control siRNA (n = 4 per group) (bar = 1 cm). ( D ) Colony formation assay was performed after transfection of Huh7 cells with miR-193a-5p Antagomir and Control (miScript Inhibitor Negative Control) (n = 4 per group) (bar = 1 cm). ( E ) Representative images and quantification of BrdU + Huh7 cells transfected with miR-193a-5p mimic or Control siRNA (n = 3 per group). ( F ) Huh7 cells were transfected with miR-193a-5p for 72 h and analyzed with flow cytometry (FACS) by DAPI staining (n = 6 per group). The percentage of cells in sub-G1, G 1 , S and G2/M phases of the cell cycle are indicated. ( G ) Huh7 cells were transfected with miR-193a-5p and treated with nocodazole (100 ng/ml) for 24 h before addition of DAPI and analyzed by FACS (n = 6 per group). The percentage of cells in the G 1 , S and G2/M phases of the cell cycle are indicated. ( H ) Western Blot showing the protein expression of the indicated proteins in Huh7 cells transfected with either miR-193a-5p mimic or Control siRNA (n = 3 per group). Results are represented as mean ± SEM. * p
    Figure Legend Snippet: Overexpression of miR-193a-5p suppresses the progression of cell cycle in vitro . ( A ) Huh7 cells were transfected with miR-193a-5p mimic and Control siRNA and cell viability was analyzed by CCK-8 assay (n = 4 per group). ( B ) Representative immunofluorescence images and quantification of Ki67 staining of Huh7 cells transfected with miR-193a-5p mimic or Control siRNA (n = 3 per group) (bar = 50 μm). ( C ) Colony formation assay was performed after transfection of Huh7 cells with miR-193a-5p mimic or Control siRNA (n = 4 per group) (bar = 1 cm). ( D ) Colony formation assay was performed after transfection of Huh7 cells with miR-193a-5p Antagomir and Control (miScript Inhibitor Negative Control) (n = 4 per group) (bar = 1 cm). ( E ) Representative images and quantification of BrdU + Huh7 cells transfected with miR-193a-5p mimic or Control siRNA (n = 3 per group). ( F ) Huh7 cells were transfected with miR-193a-5p for 72 h and analyzed with flow cytometry (FACS) by DAPI staining (n = 6 per group). The percentage of cells in sub-G1, G 1 , S and G2/M phases of the cell cycle are indicated. ( G ) Huh7 cells were transfected with miR-193a-5p and treated with nocodazole (100 ng/ml) for 24 h before addition of DAPI and analyzed by FACS (n = 6 per group). The percentage of cells in the G 1 , S and G2/M phases of the cell cycle are indicated. ( H ) Western Blot showing the protein expression of the indicated proteins in Huh7 cells transfected with either miR-193a-5p mimic or Control siRNA (n = 3 per group). Results are represented as mean ± SEM. * p

    Techniques Used: Over Expression, In Vitro, Transfection, CCK-8 Assay, Immunofluorescence, Staining, Colony Assay, Negative Control, Flow Cytometry, Cytometry, FACS, Western Blot, Expressing

    11) Product Images from "Effect of axonal micro-tubules on the morphology of retinal nerve fibers studied by second-harmonic generation"

    Article Title: Effect of axonal micro-tubules on the morphology of retinal nerve fibers studied by second-harmonic generation

    Journal: Journal of Biomedical Optics

    doi: 10.1117/1.JBO.17.11.110502

    Time-lapse SHGM of the RNFL during continuous treatment with nocodazole. (a) Thickness of the RNFL at T = 0 min. (b) Axial cross section corresponding to the dashed line in (a). From top to bottom, at 0, 20, 40, and 60 min in treatment, respectively. The length of axis labels is 30 μ m. (c) Superposed margins of the retinal nerve fibers.
    Figure Legend Snippet: Time-lapse SHGM of the RNFL during continuous treatment with nocodazole. (a) Thickness of the RNFL at T = 0 min. (b) Axial cross section corresponding to the dashed line in (a). From top to bottom, at 0, 20, 40, and 60 min in treatment, respectively. The length of axis labels is 30 μ m. (c) Superposed margins of the retinal nerve fibers.

    Techniques Used:

    RNFL thickness and SHG intensity during nocodazole treatment ( N = 4).
    Figure Legend Snippet: RNFL thickness and SHG intensity during nocodazole treatment ( N = 4).

    Techniques Used:

    12) Product Images from "Histone Deacetylase 8 Is Required for Centrosome Cohesion and Influenza A Virus Entry"

    Article Title: Histone Deacetylase 8 Is Required for Centrosome Cohesion and Influenza A Virus Entry

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002316

    HDAC8 is required for LE/LY motility. Particle tracking analysis of endocytosed EGF. (A) Occurrence of MT-dependent directed motion (DM) and their velocity (µm/s), and (B) duration (sec) in control (All*Neg), HDAC8-depleted (si HDAC8) A549 cells and cells treated with 30 µM nocodazole for 45 min (All*Neg+nocod). (A) Videos were acquired with a Visitech Spinning Disk Confocal microscope using a 100× Objective (2000 frames, Δt = 30.53 msec), within a window of 15–30 min following uptake of EGF-AF594 (1 ng/ml). Cells were transfected with a plasmid encoding NES-2×EGFP 20 h before imaging, in order to identify the cytoplasm. EGF trajectories were extracted and classified into MT-dependent DM (red) and other types of motility (blue), as shown in the insets of panel A. DM was detected in 322 segments (comprising of 11578 steps: 4.5% of total of 259821 steps), 109 segments (3235 steps: 1.06% of 305842), 37 segments (1070 steps: 0.33% of 325241) in All*Neg (n = 6 videos), HDAC8-depleted (n = 8 videos), and nocodazole treated (n = 8 videos) samples, respectively. A total of 30–50 cells were analyzed for each condition.
    Figure Legend Snippet: HDAC8 is required for LE/LY motility. Particle tracking analysis of endocytosed EGF. (A) Occurrence of MT-dependent directed motion (DM) and their velocity (µm/s), and (B) duration (sec) in control (All*Neg), HDAC8-depleted (si HDAC8) A549 cells and cells treated with 30 µM nocodazole for 45 min (All*Neg+nocod). (A) Videos were acquired with a Visitech Spinning Disk Confocal microscope using a 100× Objective (2000 frames, Δt = 30.53 msec), within a window of 15–30 min following uptake of EGF-AF594 (1 ng/ml). Cells were transfected with a plasmid encoding NES-2×EGFP 20 h before imaging, in order to identify the cytoplasm. EGF trajectories were extracted and classified into MT-dependent DM (red) and other types of motility (blue), as shown in the insets of panel A. DM was detected in 322 segments (comprising of 11578 steps: 4.5% of total of 259821 steps), 109 segments (3235 steps: 1.06% of 305842), 37 segments (1070 steps: 0.33% of 325241) in All*Neg (n = 6 videos), HDAC8-depleted (n = 8 videos), and nocodazole treated (n = 8 videos) samples, respectively. A total of 30–50 cells were analyzed for each condition.

    Techniques Used: Size-exclusion Chromatography, Microscopy, Transfection, Plasmid Preparation, Imaging

    Intact MTs are required for efficient IAV X31 infection. (A) Effect of nocodazole on IAV X31 entry. A549 cells were pretreated with 30 µM nocodazole or dmso for 30 min. Virus endocytosis assay (30 min post uptake) and HA acidification assay (1 h) was performed in the presence of drug. Data are represented as mean ± SEM. (B) Washout assay of MT perturbants. A549 cells were pretreated with 30 µM nocodazole, 50 nM taxol or dmso for 30 min. Cells were bound with virus for 30 min at 4°C, washed, and warmed in the presence of the drug for 15, 30, 45 min and 1, 2, 3, 4 h, after which the medium was replaced with medium buffered to pH 7.4 containing 20 mM NH 4 Cl to block endosome acidification. Infection was analyzed at 12 h. Data are shown as mean ± SEM. (C) Live imaging of IAV X31 particles. WGA-AF647 (5 µg/ml) (shown in green pseudocolor) and R18-labeled virus (red) were bound to control (All*Neg), HDAC8-depleted (si HDAC8) A549 cells for 30 min at 4°C. After washing, cells were warmed and imaged 3 h later with a 20× objective (see also Video S1 ). Individual and clustered X31 particles are shown as black circles. Cell border and nucleus are indicated by dotted white lines. (D) HA acidification occurs in Rab7/LAMP1-positive LEs. Control (All*Neg), HDAC8-depleted (si HDAC8) A549 cells were transfected at 48 h after depletion with plasmids expressing Rab7-EGFP and LAMP1-mCherry. HA acidification assay was performed 24 h after the plasmid transfection. Insets are magnified and shown on the right. Blue (All*Neg) and yellow arrowheads (si HDAC8) indicate LEs positive for acidified HA.
    Figure Legend Snippet: Intact MTs are required for efficient IAV X31 infection. (A) Effect of nocodazole on IAV X31 entry. A549 cells were pretreated with 30 µM nocodazole or dmso for 30 min. Virus endocytosis assay (30 min post uptake) and HA acidification assay (1 h) was performed in the presence of drug. Data are represented as mean ± SEM. (B) Washout assay of MT perturbants. A549 cells were pretreated with 30 µM nocodazole, 50 nM taxol or dmso for 30 min. Cells were bound with virus for 30 min at 4°C, washed, and warmed in the presence of the drug for 15, 30, 45 min and 1, 2, 3, 4 h, after which the medium was replaced with medium buffered to pH 7.4 containing 20 mM NH 4 Cl to block endosome acidification. Infection was analyzed at 12 h. Data are shown as mean ± SEM. (C) Live imaging of IAV X31 particles. WGA-AF647 (5 µg/ml) (shown in green pseudocolor) and R18-labeled virus (red) were bound to control (All*Neg), HDAC8-depleted (si HDAC8) A549 cells for 30 min at 4°C. After washing, cells were warmed and imaged 3 h later with a 20× objective (see also Video S1 ). Individual and clustered X31 particles are shown as black circles. Cell border and nucleus are indicated by dotted white lines. (D) HA acidification occurs in Rab7/LAMP1-positive LEs. Control (All*Neg), HDAC8-depleted (si HDAC8) A549 cells were transfected at 48 h after depletion with plasmids expressing Rab7-EGFP and LAMP1-mCherry. HA acidification assay was performed 24 h after the plasmid transfection. Insets are magnified and shown on the right. Blue (All*Neg) and yellow arrowheads (si HDAC8) indicate LEs positive for acidified HA.

    Techniques Used: Infection, Endocytosis Assay, Blocking Assay, Imaging, Whole Genome Amplification, Labeling, Transfection, Expressing, Plasmid Preparation

    HDAC8 depletion induces dispersal of LE/Lys. (A) Localization of Golgi complex (giantin, red) and LE/LYs (LAMP1, green) in control (All*Neg), HDAC8-depleted (si HDAC8) and HDAC1-depleted (si HDAC1) A549 cells. Cells were fixed and stained by indirect IFA with anti-giantin and anti-LAMP1 antibodies. Nuclei were stained with DRAQ5. (B) Determination of LE/LY dispersal index. A549 cells were depleted of HDAC1, 8, KIFC1, KIFC2, and dynactin subunits ACTR10 and DCTN2. Cells were fixed and stained by indirect IFA with anti-LAMP1 antibody. Actin was stained by phalloidin, and DNA with Hoechst. Images were acquired automatically using a 20× objective and analyzed to derive a dispersal index of LAMP1-positive endosomes. Dispersal indices were set to zero for control (All*Neg) cells, and to 1.0 for cells treated with 30 µM nocodazole for 1 h (All*Neg+nocod). Data are represented as mean ± SEM, except for KIFC1, KIFC2, ACTR10, for which the mean from 4 different siRNAs are shown (2 independent experiments). (C) EGF degradation. A549 cells were depleted of HDAC1, 8 in 96-well Matrix plates, and 48 h after depletion, starved in serum-free medium for 24 h. EGF-AF647 (200 ng/ml) was bound for 30 min on ice. After washing, the cells were warmed to 37°C in serum-containing medium to allow internalization of EGF. At 15 min and 4 h post warming, cells were washed in acid buffer (0.1 M Glycine, pH 3) for 2 min on ice to remove extracellular EGF, washed and fixed, and stained with Hoechst. EGF and nuclei were imaged automatically using a 10× objective. EGF signal intensity was quantified using ImageJ and the percentage of EGF degradation at 4 h (compared to 15 min) per nucleus is shown. Statistical significance was assessed by Student's t-test (*) p
    Figure Legend Snippet: HDAC8 depletion induces dispersal of LE/Lys. (A) Localization of Golgi complex (giantin, red) and LE/LYs (LAMP1, green) in control (All*Neg), HDAC8-depleted (si HDAC8) and HDAC1-depleted (si HDAC1) A549 cells. Cells were fixed and stained by indirect IFA with anti-giantin and anti-LAMP1 antibodies. Nuclei were stained with DRAQ5. (B) Determination of LE/LY dispersal index. A549 cells were depleted of HDAC1, 8, KIFC1, KIFC2, and dynactin subunits ACTR10 and DCTN2. Cells were fixed and stained by indirect IFA with anti-LAMP1 antibody. Actin was stained by phalloidin, and DNA with Hoechst. Images were acquired automatically using a 20× objective and analyzed to derive a dispersal index of LAMP1-positive endosomes. Dispersal indices were set to zero for control (All*Neg) cells, and to 1.0 for cells treated with 30 µM nocodazole for 1 h (All*Neg+nocod). Data are represented as mean ± SEM, except for KIFC1, KIFC2, ACTR10, for which the mean from 4 different siRNAs are shown (2 independent experiments). (C) EGF degradation. A549 cells were depleted of HDAC1, 8 in 96-well Matrix plates, and 48 h after depletion, starved in serum-free medium for 24 h. EGF-AF647 (200 ng/ml) was bound for 30 min on ice. After washing, the cells were warmed to 37°C in serum-containing medium to allow internalization of EGF. At 15 min and 4 h post warming, cells were washed in acid buffer (0.1 M Glycine, pH 3) for 2 min on ice to remove extracellular EGF, washed and fixed, and stained with Hoechst. EGF and nuclei were imaged automatically using a 10× objective. EGF signal intensity was quantified using ImageJ and the percentage of EGF degradation at 4 h (compared to 15 min) per nucleus is shown. Statistical significance was assessed by Student's t-test (*) p

    Techniques Used: Staining, Immunofluorescence

    13) Product Images from "Matrix Metalloproteinases Regulate the Formation of Dendritic Spine Head Protrusions during Chemically Induced Long-Term Potentiation"

    Article Title: Matrix Metalloproteinases Regulate the Formation of Dendritic Spine Head Protrusions during Chemically Induced Long-Term Potentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063314

    Dynamic microtubules are required for the development of SHPs upon cLTP. ( A ) Dissociated hippocampal cultures were preincubated with nocodazole (200 nM, for 4 h) to block neuronal microtubule dynamics and cLTP was then induced. Column bars show the quantification of SHPs (number/µm) under cLTP and nocodazole+cLTP conditions. Suppressing MT assembly/disassembly with nocodazole significantly reduced SHP density compared with the density measured after 40 min of cLTP. ( B ) Column bars show the quantification of SHPs (number/µm) in nocodazole washout experiments. The cultures were incubated with 200 nM nocodazole for 4 h, and nocodazole was then washed out. The cultures were kept in culture medium for 3 h, and cLTP was then induced. Blocking dynamic MTs with nocodazole followed by washout restored SHPs and significantly increased SHP density compared with unstimulated controls. The histograms show the mean ± SEM. * p
    Figure Legend Snippet: Dynamic microtubules are required for the development of SHPs upon cLTP. ( A ) Dissociated hippocampal cultures were preincubated with nocodazole (200 nM, for 4 h) to block neuronal microtubule dynamics and cLTP was then induced. Column bars show the quantification of SHPs (number/µm) under cLTP and nocodazole+cLTP conditions. Suppressing MT assembly/disassembly with nocodazole significantly reduced SHP density compared with the density measured after 40 min of cLTP. ( B ) Column bars show the quantification of SHPs (number/µm) in nocodazole washout experiments. The cultures were incubated with 200 nM nocodazole for 4 h, and nocodazole was then washed out. The cultures were kept in culture medium for 3 h, and cLTP was then induced. Blocking dynamic MTs with nocodazole followed by washout restored SHPs and significantly increased SHP density compared with unstimulated controls. The histograms show the mean ± SEM. * p

    Techniques Used: Blocking Assay, Incubation

    14) Product Images from "Primary Cilia Are Dysfunctional in Obese Adipose-Derived Mesenchymal Stem Cells"

    Article Title: Primary Cilia Are Dysfunctional in Obese Adipose-Derived Mesenchymal Stem Cells

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2017.12.022

    Less Dynamic Axoneme and Enhanced Deciliation Genes in ob-ASCs (A and B) Visceral (A) and subcutaneous ln-ASCs and ob-ASCs (B) were stained for evaluating the fluorescence intensity of axonemal acetylated microtubules (green channel) from the axonemal base to the distal tip, normalized to the corresponding cilium length (Arl13b staining, red channel). Representatives are shown. Insets depict the staining of acetylated α-tubulin (right top) and pericentrin/Arl13b (right bottom). Scale bars: 3 μm. (C and D) The fluorescence intensities of visceral (C) and subcutaneous (D) axonemal acetylated microtubules are shown. The data are from three experiments (n = 30 cilia for each condition) and presented as mean ± SEM. (E–G) ASCs were treated with 10 μM nocodazole (noc.) for 30 min and stained as indicated. Representatives are shown (E). Scale bar: 7.5 μm. Inset scale bar: 3 μm. The length of acetylated α-tubulin-labeled axonemes was evaluated. The results are based on three experiments with ASCs from three lean and three obese donors (n = 97–108 cilia for each time point) in visceral (F) and subcutaneous ASCs (G). (H and I) ASCs were cold treated for 30 min and stained for evaluating the length of acetylated α-tubulin-labeled axonemes. The data are based on three experiments with ASCs from three lean and three obese donors (n = 89–96 cilia for each time point). The cilium length is shown for visceral (H) and subcutaneous ASCs (I). (J) The gene levels of deciliation molecules. The data are based on three experiments and presented as mean ± SEM. RQ, relative quantification of gene expression. Box and whisker plots were used to show the median and the minimal to maximal range of the values in (F)–(I). Unpaired Mann-Whitney U test for (C), (D), and (F)–(I). Student's t test for (J). ∗ p
    Figure Legend Snippet: Less Dynamic Axoneme and Enhanced Deciliation Genes in ob-ASCs (A and B) Visceral (A) and subcutaneous ln-ASCs and ob-ASCs (B) were stained for evaluating the fluorescence intensity of axonemal acetylated microtubules (green channel) from the axonemal base to the distal tip, normalized to the corresponding cilium length (Arl13b staining, red channel). Representatives are shown. Insets depict the staining of acetylated α-tubulin (right top) and pericentrin/Arl13b (right bottom). Scale bars: 3 μm. (C and D) The fluorescence intensities of visceral (C) and subcutaneous (D) axonemal acetylated microtubules are shown. The data are from three experiments (n = 30 cilia for each condition) and presented as mean ± SEM. (E–G) ASCs were treated with 10 μM nocodazole (noc.) for 30 min and stained as indicated. Representatives are shown (E). Scale bar: 7.5 μm. Inset scale bar: 3 μm. The length of acetylated α-tubulin-labeled axonemes was evaluated. The results are based on three experiments with ASCs from three lean and three obese donors (n = 97–108 cilia for each time point) in visceral (F) and subcutaneous ASCs (G). (H and I) ASCs were cold treated for 30 min and stained for evaluating the length of acetylated α-tubulin-labeled axonemes. The data are based on three experiments with ASCs from three lean and three obese donors (n = 89–96 cilia for each time point). The cilium length is shown for visceral (H) and subcutaneous ASCs (I). (J) The gene levels of deciliation molecules. The data are based on three experiments and presented as mean ± SEM. RQ, relative quantification of gene expression. Box and whisker plots were used to show the median and the minimal to maximal range of the values in (F)–(I). Unpaired Mann-Whitney U test for (C), (D), and (F)–(I). Student's t test for (J). ∗ p

    Techniques Used: Staining, Fluorescence, Labeling, Expressing, Whisker Assay, MANN-WHITNEY

    15) Product Images from "Klebsiella pneumoniae Translocates across the Intestinal Epithelium via Rho GTPase- and Phosphatidylinositol 3-Kinase/Akt-Dependent Cell Invasion"

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

    Journal: Infection and Immunity

    doi: 10.1128/IAI.02345-14

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

    Techniques Used:

    16) Product Images from "Self-Assembly of the RZZ Complex into Filaments Drives Kinetochore Expansion in the Absence of Microtubule Attachment"

    Article Title: Self-Assembly of the RZZ Complex into Filaments Drives Kinetochore Expansion in the Absence of Microtubule Attachment

    Journal: Current Biology

    doi: 10.1016/j.cub.2018.08.056

    C. elegans ROD-1 Is Capable of Self-Assembly into Micrometer-Scale Filaments In Vivo (A) (Left) Schematic of experimental protocol to visualize kinetochore expansion in C. elegans early embryos. RNAi-mediated depletion of PERM-1 permeabilizes the eggshell of embryos, which are subsequently isolated from hermaphrodite adults, treated with nocodazole, and immunostained for ROD-1 and the centromere marker GFP::HCP-3 CENP-A . (Right) Immunofluorescence images of mitotic embryonic cells with and without nocodazole treatment. Arrows point at filamentous kinetochore expansions containing ROD-1 that form in the absence of microtubules. Scale bars, 2 μm. (B) (Left) Schematic of the C. elegans ). Dashed lines mark cell boundaries. Scale bars, 5 μm; blow-up, 2 μm. ). mCherry::histone H2B labels chromosomes. Filaments, typically several micrometers in length, form in the nucleus before NEBD and segregate to daughter cells by clustering at spindle poles. Kinetochore-localized GFP::ROD-1 is also visible (arrows). Time point 0 refers to the last frame before the appearance of GFP::ROD-1 on filaments and kinetochores. Scale bar, 2 μm. (D) (Left) Schematic highlighting the increase in nuclear GFP::ROD-1 levels during early embryonic development. (Right) Selected images of nuclei from a time-lapse sequence of a developing embryo expressing GFP::ROD-1 that was followed from the two-cell stage to the eight-cell stage. (Top) Images show the EMS cell in the four-cell embryo, which gives rise to the MS cell in the eight-cell embryo. (Bottom) Likewise, the ABp cell gives rise to the ABpl cell. In both instances, nuclear GFP::ROD-1 levels increase gradually during the cell cycle and are significantly higher in daughter cells. Time point 0 denotes the last frame before GFP::ROD-1 appears on kinetochores (EMS and ABp) or filaments (MS and ABpl). Similar results were obtained for nuclei of the P lineage (not shown). Scale bar, 2 μm. (E) Quantification of average GFP::ROD-1 signal in nuclei and the cytoplasm in developing embryos as shown in (D). Average fluorescence intensity was determined in images acquired every 20 s, averaged for the indicated number n of cells from at least 8 embryos, and plotted against time. Time point 0 denotes the last frame before the appearance of GFP::ROD-1 on filaments and/or kinetochores. Values are shown as mean ± 95% confidence interval for nuclear signal and as the mean for cytoplasmic signal. (F) (Left) Quantification of nuclear GFP::ROD-1 levels in cells at different developmental stages. Measurements correspond to the last frame before GFP::ROD-1 appears on filaments and/or kinetochores (time point 0 s), showing a significant increase of nuclear signal at the eight-cell stage. Mild rod-1(RNAi) was used to reduce GFP::ROD-1 levels. Values are shown as mean ± 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ∗∗∗∗ p
    Figure Legend Snippet: C. elegans ROD-1 Is Capable of Self-Assembly into Micrometer-Scale Filaments In Vivo (A) (Left) Schematic of experimental protocol to visualize kinetochore expansion in C. elegans early embryos. RNAi-mediated depletion of PERM-1 permeabilizes the eggshell of embryos, which are subsequently isolated from hermaphrodite adults, treated with nocodazole, and immunostained for ROD-1 and the centromere marker GFP::HCP-3 CENP-A . (Right) Immunofluorescence images of mitotic embryonic cells with and without nocodazole treatment. Arrows point at filamentous kinetochore expansions containing ROD-1 that form in the absence of microtubules. Scale bars, 2 μm. (B) (Left) Schematic of the C. elegans ). Dashed lines mark cell boundaries. Scale bars, 5 μm; blow-up, 2 μm. ). mCherry::histone H2B labels chromosomes. Filaments, typically several micrometers in length, form in the nucleus before NEBD and segregate to daughter cells by clustering at spindle poles. Kinetochore-localized GFP::ROD-1 is also visible (arrows). Time point 0 refers to the last frame before the appearance of GFP::ROD-1 on filaments and kinetochores. Scale bar, 2 μm. (D) (Left) Schematic highlighting the increase in nuclear GFP::ROD-1 levels during early embryonic development. (Right) Selected images of nuclei from a time-lapse sequence of a developing embryo expressing GFP::ROD-1 that was followed from the two-cell stage to the eight-cell stage. (Top) Images show the EMS cell in the four-cell embryo, which gives rise to the MS cell in the eight-cell embryo. (Bottom) Likewise, the ABp cell gives rise to the ABpl cell. In both instances, nuclear GFP::ROD-1 levels increase gradually during the cell cycle and are significantly higher in daughter cells. Time point 0 denotes the last frame before GFP::ROD-1 appears on kinetochores (EMS and ABp) or filaments (MS and ABpl). Similar results were obtained for nuclei of the P lineage (not shown). Scale bar, 2 μm. (E) Quantification of average GFP::ROD-1 signal in nuclei and the cytoplasm in developing embryos as shown in (D). Average fluorescence intensity was determined in images acquired every 20 s, averaged for the indicated number n of cells from at least 8 embryos, and plotted against time. Time point 0 denotes the last frame before the appearance of GFP::ROD-1 on filaments and/or kinetochores. Values are shown as mean ± 95% confidence interval for nuclear signal and as the mean for cytoplasmic signal. (F) (Left) Quantification of nuclear GFP::ROD-1 levels in cells at different developmental stages. Measurements correspond to the last frame before GFP::ROD-1 appears on filaments and/or kinetochores (time point 0 s), showing a significant increase of nuclear signal at the eight-cell stage. Mild rod-1(RNAi) was used to reduce GFP::ROD-1 levels. Values are shown as mean ± 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ∗∗∗∗ p

    Techniques Used: In Vivo, Isolation, Marker, Immunofluorescence, Sequencing, Expressing, Mass Spectrometry, Fluorescence

    Kinetochore Expansion Requires the RZZ Complex and SPDL1 but Is Independent of Dynein-Dynactin (A) Cartoon showing the crescent shape characteristic of the expanded outer kinetochore, which encircles the compact inner kinetochore. Components analyzed in this figure are listed on the right. (B) Immunofluorescence images showing that kinetochore expansion in nocodazole is inhibited after RNAi-mediated depletion of ROD or SPDL1. Scale bars, 5 μm; blow-ups, 1 μm. (C–F) Quantification of relative kinetochore volume for CENP-E (C) and CENP-F (E) and kinetochore signal intensity for CENP-E (D) and CENP-F (F), based on fluorescence measurements in images as shown in (B). For each condition, the mean value per kinetochore was determined for individual cells. Final values are shown as the mean of mean (n = 20 cells), normalized to the control. Error bars denote the 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. ∗∗∗∗ p
    Figure Legend Snippet: Kinetochore Expansion Requires the RZZ Complex and SPDL1 but Is Independent of Dynein-Dynactin (A) Cartoon showing the crescent shape characteristic of the expanded outer kinetochore, which encircles the compact inner kinetochore. Components analyzed in this figure are listed on the right. (B) Immunofluorescence images showing that kinetochore expansion in nocodazole is inhibited after RNAi-mediated depletion of ROD or SPDL1. Scale bars, 5 μm; blow-ups, 1 μm. (C–F) Quantification of relative kinetochore volume for CENP-E (C) and CENP-F (E) and kinetochore signal intensity for CENP-E (D) and CENP-F (F), based on fluorescence measurements in images as shown in (B). For each condition, the mean value per kinetochore was determined for individual cells. Final values are shown as the mean of mean (n = 20 cells), normalized to the control. Error bars denote the 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. ∗∗∗∗ p

    Techniques Used: Immunofluorescence, Fluorescence

    17) Product Images from "Nakiterpiosin Targets Tubulin and Triggers Mitotic Catastrophe in Human Cancer Cells"

    Article Title: Nakiterpiosin Targets Tubulin and Triggers Mitotic Catastrophe in Human Cancer Cells

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-10-0305

    Chemical structures of nakiterpiosin (NAK), nocodazole (NOC) and paclitaxel (PTX).
    Figure Legend Snippet: Chemical structures of nakiterpiosin (NAK), nocodazole (NOC) and paclitaxel (PTX).

    Techniques Used:

    18) Product Images from "The NOXA–MCL1–BIM axis defines lifespan on extended mitotic arrest"

    Article Title: The NOXA–MCL1–BIM axis defines lifespan on extended mitotic arrest

    Journal: Nature Communications

    doi: 10.1038/ncomms7891

    MCL1 undergoes degradation during M-arrest while BCL2 and BCLX remain active. ( a ) Cells were asynchronous and left untreated (Asynch), asynchronous and treated with staurosporine (STS) or synchronized by double-thymidine block and released from the arrest into DMSO-, Taxol-, Nocodazole- or BI2536-containing media. Cells were harvested in G2 and early M phase. Part of the mitotic cells were re-seeded in the presence of the above-mentioned compounds to be harvested 5 or 10 h later. Cells were harvested, lysed and analysed by immunoblotting. ( b ) Asynchronous cells were treated with DMSO or synchronized with single thymidine arrest, washed and released into fresh medium in the presence of the indicated drugs and harvested after either 8 or 12 h of M arrest. Cells of the indicated genotypes were stained using the indicated conformation-specific antibodies and analysed by flow cytometry. All cell lines were pre-treated with 200ng ml −1 doxycycline for 24h. Histograms are representatives from one out of two experiments yielding similar results.
    Figure Legend Snippet: MCL1 undergoes degradation during M-arrest while BCL2 and BCLX remain active. ( a ) Cells were asynchronous and left untreated (Asynch), asynchronous and treated with staurosporine (STS) or synchronized by double-thymidine block and released from the arrest into DMSO-, Taxol-, Nocodazole- or BI2536-containing media. Cells were harvested in G2 and early M phase. Part of the mitotic cells were re-seeded in the presence of the above-mentioned compounds to be harvested 5 or 10 h later. Cells were harvested, lysed and analysed by immunoblotting. ( b ) Asynchronous cells were treated with DMSO or synchronized with single thymidine arrest, washed and released into fresh medium in the presence of the indicated drugs and harvested after either 8 or 12 h of M arrest. Cells of the indicated genotypes were stained using the indicated conformation-specific antibodies and analysed by flow cytometry. All cell lines were pre-treated with 200ng ml −1 doxycycline for 24h. Histograms are representatives from one out of two experiments yielding similar results.

    Techniques Used: Blocking Assay, Staining, Flow Cytometry, Cytometry

    BAK is more critical than BAX for cell death induction on M-arrest in HeLa cells. ( a ) Cell fate profiles of individual cells subjected to DMSO, Nocodazole or BI2536 after transfection with the indicated siRNAs. Time in hours is indicated. ( b ) Box (interquartile range) and whisker (min to max) plots showing the elapsed time (h) between nuclear envelope breakdown (NEBD) and mitotic death for individual cells after treatments after siRNA depletion. The fraction (X/50 events) of cells undergoing the fate of interest is indicated. * P
    Figure Legend Snippet: BAK is more critical than BAX for cell death induction on M-arrest in HeLa cells. ( a ) Cell fate profiles of individual cells subjected to DMSO, Nocodazole or BI2536 after transfection with the indicated siRNAs. Time in hours is indicated. ( b ) Box (interquartile range) and whisker (min to max) plots showing the elapsed time (h) between nuclear envelope breakdown (NEBD) and mitotic death for individual cells after treatments after siRNA depletion. The fraction (X/50 events) of cells undergoing the fate of interest is indicated. * P

    Techniques Used: Transfection, Whisker Assay

    NOXA controls the mitotic lifespan by promoting MCL1 degradation. ( a ) HeLaS3 cells were synchronized and treated as described in Fig. 3 and processed for immunoblotting to evaluate NOXA expression. ( b ) Cells were transfected with the indicated siRNAs and either left untreated (Asynch.) or synchronized by double-thymidine block and harvested after the release in G2. Cells were analysed by immunoblotting ( c ) Fate profiles of individual HeLaS3-H2B–GFP cells subjected to treatment with DMSO, Nocodazole or BI2536 after transfection with the indicated siRNAs. Time in hours is indicated ( d ) Box (interquartile range) and whisker (min to max) plots showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and cell death for individual cells traced in c . The fraction (X/50 events) of cells undergoing the fate of interest is indicated. ** P
    Figure Legend Snippet: NOXA controls the mitotic lifespan by promoting MCL1 degradation. ( a ) HeLaS3 cells were synchronized and treated as described in Fig. 3 and processed for immunoblotting to evaluate NOXA expression. ( b ) Cells were transfected with the indicated siRNAs and either left untreated (Asynch.) or synchronized by double-thymidine block and harvested after the release in G2. Cells were analysed by immunoblotting ( c ) Fate profiles of individual HeLaS3-H2B–GFP cells subjected to treatment with DMSO, Nocodazole or BI2536 after transfection with the indicated siRNAs. Time in hours is indicated ( d ) Box (interquartile range) and whisker (min to max) plots showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and cell death for individual cells traced in c . The fraction (X/50 events) of cells undergoing the fate of interest is indicated. ** P

    Techniques Used: Expressing, Transfection, Blocking Assay, Whisker Assay

    BCL2 phosphorylation in M barely inhibits its activity. ( a ) HeLa-FlpIN cells transgenic for BCL2-WT, BCL2-3A and BCL2-4 A were treated with 0, 4 and 200 ng ml −1 doxycyclin (DXC) for 24 h and were either left untreated (Mito sync −) or subjected to single thymidine arrest followed by release into Nocodazole followed by mitotic shake-off (Mito sync +). Lysates were analysed by immunoblotting using the indicated antibodies. For BCL2, a short and a long exposure are displayed. ( b ) HeLa-FlpIN-parental, BCL2-WT and BCL2-4A transgenic cells were treated with the indicated amounts of doxycyclin for 24 h, and subjected to either harvesting and immunoblot analysis or ( c ) time-lapse video microscopy on treatment with Nocodazole. Fate profiles of individual cells are displayed. Time in hours is indicated.
    Figure Legend Snippet: BCL2 phosphorylation in M barely inhibits its activity. ( a ) HeLa-FlpIN cells transgenic for BCL2-WT, BCL2-3A and BCL2-4 A were treated with 0, 4 and 200 ng ml −1 doxycyclin (DXC) for 24 h and were either left untreated (Mito sync −) or subjected to single thymidine arrest followed by release into Nocodazole followed by mitotic shake-off (Mito sync +). Lysates were analysed by immunoblotting using the indicated antibodies. For BCL2, a short and a long exposure are displayed. ( b ) HeLa-FlpIN-parental, BCL2-WT and BCL2-4A transgenic cells were treated with the indicated amounts of doxycyclin for 24 h, and subjected to either harvesting and immunoblot analysis or ( c ) time-lapse video microscopy on treatment with Nocodazole. Fate profiles of individual cells are displayed. Time in hours is indicated.

    Techniques Used: Activity Assay, Transgenic Assay, Microscopy

    NOXA and BIM synergize in promoting mitotic cell death. ( a ) HeLaS3-H2B–GFP cells were transfected with a fixed amount of total siRNA using the indicated oligonucleotides and subjected to live cell imaging. Fate profiles of individual cells subjected DMSO treatment. Time is indicated in hours. ( b ) Box (interquartile range) and whisker (min to max) plots showing the elapsed time (h) between nuclear envelope breakdown (NEBD) and cell death for individual cells on the siRNA transfection in combination with Nocodazole, BI2536 or Taxol treatment. ** P
    Figure Legend Snippet: NOXA and BIM synergize in promoting mitotic cell death. ( a ) HeLaS3-H2B–GFP cells were transfected with a fixed amount of total siRNA using the indicated oligonucleotides and subjected to live cell imaging. Fate profiles of individual cells subjected DMSO treatment. Time is indicated in hours. ( b ) Box (interquartile range) and whisker (min to max) plots showing the elapsed time (h) between nuclear envelope breakdown (NEBD) and cell death for individual cells on the siRNA transfection in combination with Nocodazole, BI2536 or Taxol treatment. ** P

    Techniques Used: Transfection, Live Cell Imaging, Whisker Assay

    Activation of mitochondrial apoptosis on chronic SAC activation in HeLa cells. ( a ) Movie stills from HeLaS3-H2B–GFP cells subjected to time-lapse video microscopy following exposure to DMSO (upper panel), Nocodazole (central panel) and Nocodazole in combination with reversine (lower panel). The coloured line defines beginning of mitosis at nuclear envelope breakdown (NEBD) and end of the line matches the end of mitosis reflecting anaphase (green), death (black) and slippage (blue). Time in minutes is indicated. Scale bar, 10 μM. ( b ) Fate profiles of 50 HeLaS3-H2B–GFP cells exposed to DMSO, Nocodazole or BI2536 in the absence (DMSO) or presence of the pan-caspase inhibitor QVD. Colour-coding according to a . Time in hours is indicated. ( c ) Box (interquartile range) and whisker (min to max) plots showing the elapsed time (h) between NEBD and the indicated fate for individual cells after treatment. The fraction (X/50 events) of cells undergoing the fate of interest is indicated. Noc, Nocodazole; Q, QVD; BI, BI2536. ** P
    Figure Legend Snippet: Activation of mitochondrial apoptosis on chronic SAC activation in HeLa cells. ( a ) Movie stills from HeLaS3-H2B–GFP cells subjected to time-lapse video microscopy following exposure to DMSO (upper panel), Nocodazole (central panel) and Nocodazole in combination with reversine (lower panel). The coloured line defines beginning of mitosis at nuclear envelope breakdown (NEBD) and end of the line matches the end of mitosis reflecting anaphase (green), death (black) and slippage (blue). Time in minutes is indicated. Scale bar, 10 μM. ( b ) Fate profiles of 50 HeLaS3-H2B–GFP cells exposed to DMSO, Nocodazole or BI2536 in the absence (DMSO) or presence of the pan-caspase inhibitor QVD. Colour-coding according to a . Time in hours is indicated. ( c ) Box (interquartile range) and whisker (min to max) plots showing the elapsed time (h) between NEBD and the indicated fate for individual cells after treatment. The fraction (X/50 events) of cells undergoing the fate of interest is indicated. Noc, Nocodazole; Q, QVD; BI, BI2536. ** P

    Techniques Used: Activation Assay, Microscopy, Whisker Assay

    Noxa and Bim synergize in promoting mitotic death in A549 cells. ( a ) A549 cells were transfected with the indicated siRNAs, synchronized with double-thymidine block and released into medium containing Nocodazole. Cells were harvested after increasing times of mitotic arrest and processed for immunoblotting with the indicated antibodies. ( b , c ) A549-H2B-mRFP cells were transfected with the indicated siRNAs, synchronized, subjected to treatment with DMSO, Nocodazole or Nocodazole and ABT-737 and analysed by live cell imaging. ( b ) Box (interquartile range) and whisker (min to max) plots showing the elapsed time (h) between nuclear envelope breakdown (NEBD) and cell death for individual cells. ** P
    Figure Legend Snippet: Noxa and Bim synergize in promoting mitotic death in A549 cells. ( a ) A549 cells were transfected with the indicated siRNAs, synchronized with double-thymidine block and released into medium containing Nocodazole. Cells were harvested after increasing times of mitotic arrest and processed for immunoblotting with the indicated antibodies. ( b , c ) A549-H2B-mRFP cells were transfected with the indicated siRNAs, synchronized, subjected to treatment with DMSO, Nocodazole or Nocodazole and ABT-737 and analysed by live cell imaging. ( b ) Box (interquartile range) and whisker (min to max) plots showing the elapsed time (h) between nuclear envelope breakdown (NEBD) and cell death for individual cells. ** P

    Techniques Used: Transfection, Blocking Assay, Live Cell Imaging, Whisker Assay

    19) Product Images from "Visualizing Mutation-Specific Differences in the Trafficking-Deficient Phenotype of Kv11.1 Proteins Linked to Long QT Syndrome Type 2"

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

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00584

    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.
    Figure Legend 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.

    Techniques Used: Immunostaining, Expressing, Incubation, Labeling

    20) Product Images from "Human Geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis"

    Article Title: Human Geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600314

    Model for the role of human Geminin in regulating DNA replication: Geminin promotes licensing at mitotic exit and prevents rereplication during S phase. ( A ) Human Geminin depletion has different consequences in S and M phases (see Discussion for details). The diagram represents the levels of CDT1 and Geminin during the cell cycle. The two lines are independent lines that do not refer to each other. In the lower part of the panel, U2OS cells synchronized in different ways (see below) were collected at different times during cell cycle and analyzed by FACS analysis or immunoblotted for the indicated proteins. The cells were collected (left to right) 6 h after nocodazole release (lane 1), 4 h (lane 2) and 8 h (lane 3) after thymidine release or taken from the attached (lane 4) or detached (lane 5) populations of nocodazole-treated cells. ( B ) Human Geminin stabilizes CDT1 during mitosis and appears not to restrict pre-RC formation. In contrast, Geminin is required for the accumulation of CDT1. The main regulator of origin licensing is instead CDK1, which inhibits pre-RC formation by interfering with CDT1 and MCM binding to chromatin.
    Figure Legend Snippet: Model for the role of human Geminin in regulating DNA replication: Geminin promotes licensing at mitotic exit and prevents rereplication during S phase. ( A ) Human Geminin depletion has different consequences in S and M phases (see Discussion for details). The diagram represents the levels of CDT1 and Geminin during the cell cycle. The two lines are independent lines that do not refer to each other. In the lower part of the panel, U2OS cells synchronized in different ways (see below) were collected at different times during cell cycle and analyzed by FACS analysis or immunoblotted for the indicated proteins. The cells were collected (left to right) 6 h after nocodazole release (lane 1), 4 h (lane 2) and 8 h (lane 3) after thymidine release or taken from the attached (lane 4) or detached (lane 5) populations of nocodazole-treated cells. ( B ) Human Geminin stabilizes CDT1 during mitosis and appears not to restrict pre-RC formation. In contrast, Geminin is required for the accumulation of CDT1. The main regulator of origin licensing is instead CDK1, which inhibits pre-RC formation by interfering with CDT1 and MCM binding to chromatin.

    Techniques Used: FACS, Binding Assay

    Human Geminin stabilizes basal levels of CDT1 in S phase. ( A ) Experimental outline. ( B, C ) Geminin depletion in S phase leads to a decrease in CDT1 protein levels. (B) U2OS and (C) HeLa were treated during G1 with siRNAs for the indicated genes and collected during S phase 14 and 12 h after nocodazole release, respectively, and treated with DMSO or MG132 for the last 4 h of incubation. The cell lysates were immunoblotted for the indicated proteins. ( D ) GBD is located between residues 150 and 190 of human CDT1 protein. ( E, F ) Overexpression of Geminin in S phase stabilizes CDT1 protein. (E) Plasmids expressing HA-CDT1 (WT and ΔGBD) were injected, with or without an expression plasmid for Geminin, in S phase-synchronized HeLa cells and 3 h later fixed and stained for HA or DAPI. IgG were co-injected and counterstained to identify the injected cells. MG132 was used in parallel as control for protein expression. Representative fields are shown. (F) Histogram representing the percentage of cells positive for CDT1 staining in the injected population of cells for each of the treatment shown in (E).
    Figure Legend Snippet: Human Geminin stabilizes basal levels of CDT1 in S phase. ( A ) Experimental outline. ( B, C ) Geminin depletion in S phase leads to a decrease in CDT1 protein levels. (B) U2OS and (C) HeLa were treated during G1 with siRNAs for the indicated genes and collected during S phase 14 and 12 h after nocodazole release, respectively, and treated with DMSO or MG132 for the last 4 h of incubation. The cell lysates were immunoblotted for the indicated proteins. ( D ) GBD is located between residues 150 and 190 of human CDT1 protein. ( E, F ) Overexpression of Geminin in S phase stabilizes CDT1 protein. (E) Plasmids expressing HA-CDT1 (WT and ΔGBD) were injected, with or without an expression plasmid for Geminin, in S phase-synchronized HeLa cells and 3 h later fixed and stained for HA or DAPI. IgG were co-injected and counterstained to identify the injected cells. MG132 was used in parallel as control for protein expression. Representative fields are shown. (F) Histogram representing the percentage of cells positive for CDT1 staining in the injected population of cells for each of the treatment shown in (E).

    Techniques Used: Incubation, Over Expression, Expressing, Injection, Plasmid Preparation, Staining

    21) Product Images from "Dynamic GLUT4 sorting through a syntaxin-6 compartment in muscle cells is derailed by insulin resistance-causing ceramide"

    Article Title: Dynamic GLUT4 sorting through a syntaxin-6 compartment in muscle cells is derailed by insulin resistance-causing ceramide

    Journal: Biology Open

    doi: 10.1242/bio.20147898

    Nocodazole prevents both GLUT4 sorting into the Syntaxin-6-positive perinuclear sub-compartment and insulin-responsive GLUT4 re-exocytosis. (A) Cell surface GLUT4 myc was labeled at 4°C before cells were re-warmed at 37°C for 30 min with or without 3 µM nocodazole. For recovery, nocodazole was washed out after 25 min and replaced with serum free medium for indicated recovery times. Fixed cells were stained for endogenous Stx6 (green). Inset = single optical slice of the perinuclear region. Representative tubulin staining is shown for each condition (far right). (B) Quantification of the co-localization between GLUT4 myc and Stx6 using Pearson's Correlation coefficient (N = 2–4, 10–15 cells per experiment), *p
    Figure Legend Snippet: Nocodazole prevents both GLUT4 sorting into the Syntaxin-6-positive perinuclear sub-compartment and insulin-responsive GLUT4 re-exocytosis. (A) Cell surface GLUT4 myc was labeled at 4°C before cells were re-warmed at 37°C for 30 min with or without 3 µM nocodazole. For recovery, nocodazole was washed out after 25 min and replaced with serum free medium for indicated recovery times. Fixed cells were stained for endogenous Stx6 (green). Inset = single optical slice of the perinuclear region. Representative tubulin staining is shown for each condition (far right). (B) Quantification of the co-localization between GLUT4 myc and Stx6 using Pearson's Correlation coefficient (N = 2–4, 10–15 cells per experiment), *p

    Techniques Used: Labeling, Staining

    22) Product Images from "Statin-induced depletion of geranylgeranyl pyrophosphate inhibits cell proliferation by a novel pathway of Skp2 degradation"

    Article Title: Statin-induced depletion of geranylgeranyl pyrophosphate inhibits cell proliferation by a novel pathway of Skp2 degradation

    Journal: Oncotarget

    doi:

    Skp2 decline is due to blockade of geranylgeranylation (A) HeLa cells were treated with 40μM lovastatin in the presence of geranylgeranylpyrophosphate (GGPP) or farnesylpyrophosphate (FPP). After 24 hours incubation, cells were harvested and subjected to western blotting analysis. (B) HeLa cells were treated 20μM lovastatin, geranylgeranyltransferase inhibitor GGTI-298 or farnesyltransferase inhibitor FTI-277 for 36 hours and analysed by western blotting. (C) HeLa cells were treated with 10μM GGTI-298 for different times (as indicated) after which Skp2, p27 levels were determined by immunoblot analysis. (D) HeLa cells were treated with 10μM GGTI-298 in the presence and absence of 20mM MG132. After 24 hours, samples were harvested and analysed by western blotting. (E) Control MEFs and p27 −/− MEFs were treated with 10 μM GGTI-298 for 24 hours as indicated, harvested and analysed by western blotting analysis (left panel). Flow cytometry analysis of BrdU labeled cells to determine cell cycle phase distribution was performed. The percentage of cells in G1, S or G2/M phase is shown for at least three independent experiments (right panel). (F) Control MEFs and Cdh1 −/− MEFs were treated with 10μM GGTI-298 for 24 hours then harvested and subjected to western blotting analysis. (G) Hela cells were treated with 50ng/ml Nocodazole for 18 hours after which they were supplemented with GGTI-298 at a final concentration of 10μM or solvent control. After a further 3 hours cells were supplemented with MG132 (20μM) or solvent and incubated for a further 9 hours. Cells were then harvested and processed for western blotting analysis.
    Figure Legend Snippet: Skp2 decline is due to blockade of geranylgeranylation (A) HeLa cells were treated with 40μM lovastatin in the presence of geranylgeranylpyrophosphate (GGPP) or farnesylpyrophosphate (FPP). After 24 hours incubation, cells were harvested and subjected to western blotting analysis. (B) HeLa cells were treated 20μM lovastatin, geranylgeranyltransferase inhibitor GGTI-298 or farnesyltransferase inhibitor FTI-277 for 36 hours and analysed by western blotting. (C) HeLa cells were treated with 10μM GGTI-298 for different times (as indicated) after which Skp2, p27 levels were determined by immunoblot analysis. (D) HeLa cells were treated with 10μM GGTI-298 in the presence and absence of 20mM MG132. After 24 hours, samples were harvested and analysed by western blotting. (E) Control MEFs and p27 −/− MEFs were treated with 10 μM GGTI-298 for 24 hours as indicated, harvested and analysed by western blotting analysis (left panel). Flow cytometry analysis of BrdU labeled cells to determine cell cycle phase distribution was performed. The percentage of cells in G1, S or G2/M phase is shown for at least three independent experiments (right panel). (F) Control MEFs and Cdh1 −/− MEFs were treated with 10μM GGTI-298 for 24 hours then harvested and subjected to western blotting analysis. (G) Hela cells were treated with 50ng/ml Nocodazole for 18 hours after which they were supplemented with GGTI-298 at a final concentration of 10μM or solvent control. After a further 3 hours cells were supplemented with MG132 (20μM) or solvent and incubated for a further 9 hours. Cells were then harvested and processed for western blotting analysis.

    Techniques Used: Incubation, Western Blot, Flow Cytometry, Cytometry, Labeling, Concentration Assay

    23) Product Images from "Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes"

    Article Title: Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039774

    Depletion of the Arp2/3 complex increases the amount of endosomal WASH. (A) Stable 3T3 cells expressing GFP-WASH were depleted from the Arp2/3 complex using siRNAs and analyzed by Western Blot. (B) siRNA-transfected cells were treated with 10 µM nocodazole for 1 h, then processed for immunofluorescence using antibodies recognizing EEA1 and the p16Arc subunit of the Arp2/3 complex, and observed by spinning disk confocal microscopy (single planes). Scale bar: 10 µm. Arp2/3 depletion increases endosomal WASH staining. (C–D) Image stacks (36 cells, 6046 endosomes for Ctrl siRNA; 31 cells, 3889 endosomes for p34Arc #1 siRNA; 26 cells, 3470 endosomes for p34Arc #2 siRNA) were processed and presented as in Fig. 2 D–E after normalization of endosomes volumes. Upon Arp2/3 complex depletion, the intensity and ‘apparent concentration’ of GFP-WASH domains increases but does not depend on endosome volume. *: p
    Figure Legend Snippet: Depletion of the Arp2/3 complex increases the amount of endosomal WASH. (A) Stable 3T3 cells expressing GFP-WASH were depleted from the Arp2/3 complex using siRNAs and analyzed by Western Blot. (B) siRNA-transfected cells were treated with 10 µM nocodazole for 1 h, then processed for immunofluorescence using antibodies recognizing EEA1 and the p16Arc subunit of the Arp2/3 complex, and observed by spinning disk confocal microscopy (single planes). Scale bar: 10 µm. Arp2/3 depletion increases endosomal WASH staining. (C–D) Image stacks (36 cells, 6046 endosomes for Ctrl siRNA; 31 cells, 3889 endosomes for p34Arc #1 siRNA; 26 cells, 3470 endosomes for p34Arc #2 siRNA) were processed and presented as in Fig. 2 D–E after normalization of endosomes volumes. Upon Arp2/3 complex depletion, the intensity and ‘apparent concentration’ of GFP-WASH domains increases but does not depend on endosome volume. *: p

    Techniques Used: Expressing, Western Blot, Transfection, Immunofluorescence, Confocal Microscopy, Staining, Concentration Assay

    Automated quantification of the increase of endosomal WASH upon actin depolymerization. (A) 3T3 cells were loaded with fluorescent Tf until equilibrium, then treated with 10 µM nocodazole in the continuous presence of Tf for 1 h, then treated with 0.2 µM LatA or carrier in the presence of nocodazole and Tf for 10 min. Cells were then processed for immunofluorescence and observed as in Fig. 1 . Scale bar: 10 µm (1 µm in inserts). The nocodazole treatment induces scattering of the endosomes thus allowing detection of the whole population of endosomes. The LatA treatment enlarges endosomes and increases the intensity of WASH on endosomes. (B) Image processing workflow (see also Methods). WASH and Tf images were processed as in Fig. 1 . As the endosomes were scattered due to nocodazole treatment, both Tf-positive endosomes and WASH domains could be detected automatically after 3D segmentation, then each WASH domain was assigned to its proper endosome using a custom-made program (see Methods) and the WASH intensity was measured on each endosome. (C) Image stacks of cells treated as in Fig. 2 A were segmented in 3D for both channels and analyzed (24 cells, 11872 endosomes for control; 44 cells, 11633 endosomes for LatA). The LatA treatment induces a shift of endosome sizes towards larger volumes (p
    Figure Legend Snippet: Automated quantification of the increase of endosomal WASH upon actin depolymerization. (A) 3T3 cells were loaded with fluorescent Tf until equilibrium, then treated with 10 µM nocodazole in the continuous presence of Tf for 1 h, then treated with 0.2 µM LatA or carrier in the presence of nocodazole and Tf for 10 min. Cells were then processed for immunofluorescence and observed as in Fig. 1 . Scale bar: 10 µm (1 µm in inserts). The nocodazole treatment induces scattering of the endosomes thus allowing detection of the whole population of endosomes. The LatA treatment enlarges endosomes and increases the intensity of WASH on endosomes. (B) Image processing workflow (see also Methods). WASH and Tf images were processed as in Fig. 1 . As the endosomes were scattered due to nocodazole treatment, both Tf-positive endosomes and WASH domains could be detected automatically after 3D segmentation, then each WASH domain was assigned to its proper endosome using a custom-made program (see Methods) and the WASH intensity was measured on each endosome. (C) Image stacks of cells treated as in Fig. 2 A were segmented in 3D for both channels and analyzed (24 cells, 11872 endosomes for control; 44 cells, 11633 endosomes for LatA). The LatA treatment induces a shift of endosome sizes towards larger volumes (p

    Techniques Used: Immunofluorescence

    Arp2/3 depletion induces the appearance of elongated WASH domains. (A) Stable 3T3 cells expressing GFP-WASH were depleted of the Arp2/3 complex and treated with nocodazole as in Fig. 4 B. Scale bar: 1 µm. GFP-WASH forms a crescent at the surface of large EEA1 positive endosomes upon Arp2/3 depletion. (B) 3T3 cells were depleted of the Arp2/3 complex and processed for immunofluorescence using WASH and p16Arc antibodies. Scale bar: 10 µm. WASH crescents are also observed with endogenous WASH in the absence of nocodazole. (C) Stable 3T3 cells expressing GFP-WASH depleted for the Arp2/3 complex and imaged by live spinning disk confocal microscopy after Tf loading. Elapsed time is in seconds. Scale bar: 2 µm. Arp2/3 depletion inhibits Tf uptake and WASH forms crescents at the surface of large endosomes that are dimly stained by Tf.
    Figure Legend Snippet: Arp2/3 depletion induces the appearance of elongated WASH domains. (A) Stable 3T3 cells expressing GFP-WASH were depleted of the Arp2/3 complex and treated with nocodazole as in Fig. 4 B. Scale bar: 1 µm. GFP-WASH forms a crescent at the surface of large EEA1 positive endosomes upon Arp2/3 depletion. (B) 3T3 cells were depleted of the Arp2/3 complex and processed for immunofluorescence using WASH and p16Arc antibodies. Scale bar: 10 µm. WASH crescents are also observed with endogenous WASH in the absence of nocodazole. (C) Stable 3T3 cells expressing GFP-WASH depleted for the Arp2/3 complex and imaged by live spinning disk confocal microscopy after Tf loading. Elapsed time is in seconds. Scale bar: 2 µm. Arp2/3 depletion inhibits Tf uptake and WASH forms crescents at the surface of large endosomes that are dimly stained by Tf.

    Techniques Used: Expressing, Immunofluorescence, Confocal Microscopy, Staining

    Direct observation of WASH domain coalescence in live cells upon actin depolymerization. Stable 3T3 cells expressing GFP-WASH and transiently expressing mCherry-Rab5Q79L were treated with 10 µM nocodazole for 1 h, then imaged by live spinning disk confocal microscopy. 12 planes separated by an increment of 0.4 µm were acquired at each time point, and z-projected. Elapsed time is in seconds. Scale bar: 2 µm. 0.2 µM LatA was added at the beginning of the movie. Arrowheads show individual WASH domains that fuse sequentially.
    Figure Legend Snippet: Direct observation of WASH domain coalescence in live cells upon actin depolymerization. Stable 3T3 cells expressing GFP-WASH and transiently expressing mCherry-Rab5Q79L were treated with 10 µM nocodazole for 1 h, then imaged by live spinning disk confocal microscopy. 12 planes separated by an increment of 0.4 µm were acquired at each time point, and z-projected. Elapsed time is in seconds. Scale bar: 2 µm. 0.2 µM LatA was added at the beginning of the movie. Arrowheads show individual WASH domains that fuse sequentially.

    Techniques Used: Expressing, Confocal Microscopy

    24) Product Images from "Desmosomal cadherins utilize distinct kinesins for assembly into desmosomes"

    Article Title: Desmosomal cadherins utilize distinct kinesins for assembly into desmosomes

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201106057

    Desmosomal cadherins require MTs for rapid accumulation at intercellular junctions. (A) Dual-label immunofluorescence revealed that Dsg2 and Dsc2 colocalize at cell–cell junctions but are present in separate cytoplasmic vesicles. Boxes indicate areas of magnification on the right. (B) Cells coexpressing Dsg2-GFP and tubulin-mCherry (blue) were imaged at 5-s intervals ( Video 2 ). Dsg2-containing vesicles move along MTs toward the plasma membrane. (C) Contacting cells expressing Dsg2-GFP and tubulin-mCherry were imaged at 5-s intervals. Video 3 shows the cropped area in the left image. White brackets indicate cell–cell contact. Green track shows the path taken by a Dsg2-containing vesicle to the contact site. (D) Scc9s were switched to low-calcium medium (LCM) for 2 h before incubation with nocodazole for 1 h in low-calcium medium and then switched to normal Ca 2+ (with or without nocodazole) for 30 min to trigger junction assembly. Cytoplasmic Dsg2 and Dsc2 vesicles are present in control (Cntrl) and nocodazole (noc)-treated cells in low-calcium medium. Disruption of MT delays Dsg2 and Dsc2 assembly at newly forming cell–cell interfaces. Bars: (A [left] and D) 20 µm; (A [right] and C) 10 µm; (B) 5 µm.
    Figure Legend Snippet: Desmosomal cadherins require MTs for rapid accumulation at intercellular junctions. (A) Dual-label immunofluorescence revealed that Dsg2 and Dsc2 colocalize at cell–cell junctions but are present in separate cytoplasmic vesicles. Boxes indicate areas of magnification on the right. (B) Cells coexpressing Dsg2-GFP and tubulin-mCherry (blue) were imaged at 5-s intervals ( Video 2 ). Dsg2-containing vesicles move along MTs toward the plasma membrane. (C) Contacting cells expressing Dsg2-GFP and tubulin-mCherry were imaged at 5-s intervals. Video 3 shows the cropped area in the left image. White brackets indicate cell–cell contact. Green track shows the path taken by a Dsg2-containing vesicle to the contact site. (D) Scc9s were switched to low-calcium medium (LCM) for 2 h before incubation with nocodazole for 1 h in low-calcium medium and then switched to normal Ca 2+ (with or without nocodazole) for 30 min to trigger junction assembly. Cytoplasmic Dsg2 and Dsc2 vesicles are present in control (Cntrl) and nocodazole (noc)-treated cells in low-calcium medium. Disruption of MT delays Dsg2 and Dsc2 assembly at newly forming cell–cell interfaces. Bars: (A [left] and D) 20 µm; (A [right] and C) 10 µm; (B) 5 µm.

    Techniques Used: Immunofluorescence, Expressing, Laser Capture Microdissection, Incubation

    25) Product Images from "Interaction between the Type III Effector VopO and GEF-H1 Activates the RhoA-ROCK Pathway"

    Article Title: Interaction between the Type III Effector VopO and GEF-H1 Activates the RhoA-ROCK Pathway

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004694

    Identification of the stress fiber formation-inducing T3SS2 effector VopO. (A) HeLa cells were infected with POR-2, POR-2 ∆vcrD2 (a T3SS1- and T3SS2-deficient strain), POR-2 ∆vopO (a vopO mutant strain derived from POR-2), or POR-2 ∆vopO/pvopO (a strain complemented with the vopO gene) for 3 h, or were treated with 10 mM nocodazole for 1 h. After infection or nocodazole treatment, the cells were stained to detect F-actin (green) and cellular and bacterial DNA (blue). (B) G-LISA was used to evaluate the relative RhoA activation level in cells infected with isogenic V. parahaemolyticus mutant strains for 150 min or treated with 10 mM nocodazole for 30 min. The asterisks indicate results that differ significantly from those obtained using the parent strain (POR-2) (* p
    Figure Legend Snippet: Identification of the stress fiber formation-inducing T3SS2 effector VopO. (A) HeLa cells were infected with POR-2, POR-2 ∆vcrD2 (a T3SS1- and T3SS2-deficient strain), POR-2 ∆vopO (a vopO mutant strain derived from POR-2), or POR-2 ∆vopO/pvopO (a strain complemented with the vopO gene) for 3 h, or were treated with 10 mM nocodazole for 1 h. After infection or nocodazole treatment, the cells were stained to detect F-actin (green) and cellular and bacterial DNA (blue). (B) G-LISA was used to evaluate the relative RhoA activation level in cells infected with isogenic V. parahaemolyticus mutant strains for 150 min or treated with 10 mM nocodazole for 30 min. The asterisks indicate results that differ significantly from those obtained using the parent strain (POR-2) (* p

    Techniques Used: Infection, Mutagenesis, Derivative Assay, Staining, Activation Assay

    26) Product Images from "Visualization of Microtubule Growth in Cultured Neurons via the Use of EB3-GFP (End-Binding Protein 3-Green Fluorescent Protein)"

    Article Title: Visualization of Microtubule Growth in Cultured Neurons via the Use of EB3-GFP (End-Binding Protein 3-Green Fluorescent Protein)

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.23-07-02655.2003

    +TIPs in hippocampal neurons. A–K , Hippocampal neurons of 2–6 d in culture were fixed and incubated with a mixture of antibodies recognizing both CLIP-115 and CLIP-170 ( A–D ), with an antibody mixture recognizing EB1 and EB3 ( E–G ), with a mixture recognizing EB3 and β-tubulin ( H, I ), or with the single EB1 ( J ) and EB3 ( K ) antibodies. B (growth cone) and C (neurites) are magnifications of the rectangles in A and D , respectively. Examples of comet-like CLIP dashes are indicated by arrows . In G the merged image of EB1 ( E ) and EB3 ( F ) staining is shown, in which EB1 is in red and EB3 is in green . Clear comet-like labeling is visible with both antibodies in the neuron and in the two glial cells. In H (neuronal growth cone) and I (cell periphery of a glial cell) deconvolution of confocal images is used to demonstrate clearly the EB3 localization to MT distal ends. The large arrows in H and I indicate clear examples of such EB3 localization. The small arrow in I points toward a MT end, which is not labeled by EB3. Given that the other end of this small MT is stained, we presume that this end represents a MT minus end. The cell periphery of the glial cell in I is particular in that it contains several of such “free” MTs. In J and K nocodazole was added to the culture medium before fixation of the cells. Note that the typical comet-like staining of EB1 and EB3 has vanished after this brief treatment. L–Q , Hippocampal neurons, transfected with EB3-GFP, were fixed 3–4 d after plating and stained with cytoskeletal markers. Costaining of EB3-GFP ( L ) in a growth cone with antibodies against tyrosinated α-tubulin ( M ) reveals that the GFP signals are located at the tip of MTs (the arrow indicates an example of such a tip in the merged image in N ). Costaining of EB3-GFP ( O ) in a transfected neuron with antibodies against actin, followed by Alexa 594-conjugated secondary antibody incubation ( P ), reveals that EB3-GFP-positive MT plus ends are present in the growth cone and sometimes are detected in actin-rich filopodial extensions (see merged image in Q ).
    Figure Legend Snippet: +TIPs in hippocampal neurons. A–K , Hippocampal neurons of 2–6 d in culture were fixed and incubated with a mixture of antibodies recognizing both CLIP-115 and CLIP-170 ( A–D ), with an antibody mixture recognizing EB1 and EB3 ( E–G ), with a mixture recognizing EB3 and β-tubulin ( H, I ), or with the single EB1 ( J ) and EB3 ( K ) antibodies. B (growth cone) and C (neurites) are magnifications of the rectangles in A and D , respectively. Examples of comet-like CLIP dashes are indicated by arrows . In G the merged image of EB1 ( E ) and EB3 ( F ) staining is shown, in which EB1 is in red and EB3 is in green . Clear comet-like labeling is visible with both antibodies in the neuron and in the two glial cells. In H (neuronal growth cone) and I (cell periphery of a glial cell) deconvolution of confocal images is used to demonstrate clearly the EB3 localization to MT distal ends. The large arrows in H and I indicate clear examples of such EB3 localization. The small arrow in I points toward a MT end, which is not labeled by EB3. Given that the other end of this small MT is stained, we presume that this end represents a MT minus end. The cell periphery of the glial cell in I is particular in that it contains several of such “free” MTs. In J and K nocodazole was added to the culture medium before fixation of the cells. Note that the typical comet-like staining of EB1 and EB3 has vanished after this brief treatment. L–Q , Hippocampal neurons, transfected with EB3-GFP, were fixed 3–4 d after plating and stained with cytoskeletal markers. Costaining of EB3-GFP ( L ) in a growth cone with antibodies against tyrosinated α-tubulin ( M ) reveals that the GFP signals are located at the tip of MTs (the arrow indicates an example of such a tip in the merged image in N ). Costaining of EB3-GFP ( O ) in a transfected neuron with antibodies against actin, followed by Alexa 594-conjugated secondary antibody incubation ( P ), reveals that EB3-GFP-positive MT plus ends are present in the growth cone and sometimes are detected in actin-rich filopodial extensions (see merged image in Q ).

    Techniques Used: Incubation, Cross-linking Immunoprecipitation, Staining, Labeling, Transfection

    27) Product Images from "Cell Cycle-Dependent Dynamics of the Golgi-Centrosome Association in Motile Cells"

    Article Title: Cell Cycle-Dependent Dynamics of the Golgi-Centrosome Association in Motile Cells

    Journal: Cells

    doi: 10.3390/cells9051069

    Golgi stretching around the nucleus is centrosome-independent and microtubule-dependent. ( a – b’ ) Frames from a time-lapse imaging sequence of RPE1 cells stably expressing Golgi (RFP-TGN, red) and centrosome markers (centrin1-GFP, green). E mode progression over 5 h in ( a ) control cell or ( b ) cell pretreated by Centrinone B for 72 h. Scale 5 µm. Time, hours, minutes. ( c ) Quantification of PER and GG in fixed S-phase cells (BRDu-positive) pretreated with DMSO or Centrinone B for 72 h. Student t -test showed no significant differences between DMSO and Centrinone B-treated cells for either PER or GG. n = 49. Error bars, SD. Red line, mean. ( d ) Change of Golgi extension during 30 min live-cell imaging of cells with and without MTs. Cells pretreated on ice for 45 min were recorded in the presence of DMSO (control) vs. nocodazole. PER index before and after imaging is shown. n = 3. ( e – e’’ ) Localization of the Golgi (giantin, cyan) and MTs (α-tubulin, red) in E mode, immunostaining, centrin1-GFP (green). ( e ) Cell overview shown as a maximum intensity projection of entire laser scanning confocal stack (5.25 µm-thick). Scale 10 µm. ( e’ , e” ) Maximum intensity projections of dorsal (0.84 µm-thick) and ventral (0.63 µm-thick) sub-stacks of the central cell area. Scale 5 µm. ( f ) Progressive movement (white arrows) of dynamic Golgi tubule (TGN, red) along the nuclear equator shown in 20 s intervals. Asterisk denotes starting position of Golgi membrane tubule movement. Scale 5 µm. Time, minutes, seconds. ( g ) Live cell visualization of E mode progression in cells pretreated on ice for 45 min and recorded in the presence of DMSO ( g ) or nocodazole ( h , as quantified in d ). Golgi (RFP-TGN, red). Centrosome (centrin1-GFP, green). Scale 5 µm. Time, seconds. Images in ( a , b , f , g , h ) are maximum intensity projection of entire spinning disk confocal stacks.
    Figure Legend Snippet: Golgi stretching around the nucleus is centrosome-independent and microtubule-dependent. ( a – b’ ) Frames from a time-lapse imaging sequence of RPE1 cells stably expressing Golgi (RFP-TGN, red) and centrosome markers (centrin1-GFP, green). E mode progression over 5 h in ( a ) control cell or ( b ) cell pretreated by Centrinone B for 72 h. Scale 5 µm. Time, hours, minutes. ( c ) Quantification of PER and GG in fixed S-phase cells (BRDu-positive) pretreated with DMSO or Centrinone B for 72 h. Student t -test showed no significant differences between DMSO and Centrinone B-treated cells for either PER or GG. n = 49. Error bars, SD. Red line, mean. ( d ) Change of Golgi extension during 30 min live-cell imaging of cells with and without MTs. Cells pretreated on ice for 45 min were recorded in the presence of DMSO (control) vs. nocodazole. PER index before and after imaging is shown. n = 3. ( e – e’’ ) Localization of the Golgi (giantin, cyan) and MTs (α-tubulin, red) in E mode, immunostaining, centrin1-GFP (green). ( e ) Cell overview shown as a maximum intensity projection of entire laser scanning confocal stack (5.25 µm-thick). Scale 10 µm. ( e’ , e” ) Maximum intensity projections of dorsal (0.84 µm-thick) and ventral (0.63 µm-thick) sub-stacks of the central cell area. Scale 5 µm. ( f ) Progressive movement (white arrows) of dynamic Golgi tubule (TGN, red) along the nuclear equator shown in 20 s intervals. Asterisk denotes starting position of Golgi membrane tubule movement. Scale 5 µm. Time, minutes, seconds. ( g ) Live cell visualization of E mode progression in cells pretreated on ice for 45 min and recorded in the presence of DMSO ( g ) or nocodazole ( h , as quantified in d ). Golgi (RFP-TGN, red). Centrosome (centrin1-GFP, green). Scale 5 µm. Time, seconds. Images in ( a , b , f , g , h ) are maximum intensity projection of entire spinning disk confocal stacks.

    Techniques Used: Imaging, Sequencing, Stable Transfection, Expressing, Live Cell Imaging, Immunostaining

    28) Product Images from "Mitosis can drive cell cannibalism through entosis"

    Article Title: Mitosis can drive cell cannibalism through entosis

    Journal: eLife

    doi: 10.7554/eLife.27134

    Paclitaxel/taxol treatment promotes mitotic deadhesion, rounding and entosis. ( a ) Representative timelapse images of adherent 16HBE cells treated with 1 μM taxol. Cell 1 (outlined white) rounds up in prometaphase and subsequently penetrates an adherent interphase neighbour (Cell 2, outlined yellow). Timestamps are shown (hr:min) and scale bar = 10 μm. ( b ) Representative confocal/DIC images of adherent cell-in-cell structures in 16HBE treated with taxol (1 μM), nocodazole (100 ng/ml) or STLC (20 μM) for 24 hr. Cells were stained for DNA (blue), scale bar = 10 μm. ( c ) Quantification of drug-induced cell-in-cell formation. > 150 cells were counted per sample/experiment, across three separate experiments. Error bars denote mean±SEM. **p
    Figure Legend Snippet: Paclitaxel/taxol treatment promotes mitotic deadhesion, rounding and entosis. ( a ) Representative timelapse images of adherent 16HBE cells treated with 1 μM taxol. Cell 1 (outlined white) rounds up in prometaphase and subsequently penetrates an adherent interphase neighbour (Cell 2, outlined yellow). Timestamps are shown (hr:min) and scale bar = 10 μm. ( b ) Representative confocal/DIC images of adherent cell-in-cell structures in 16HBE treated with taxol (1 μM), nocodazole (100 ng/ml) or STLC (20 μM) for 24 hr. Cells were stained for DNA (blue), scale bar = 10 μm. ( c ) Quantification of drug-induced cell-in-cell formation. > 150 cells were counted per sample/experiment, across three separate experiments. Error bars denote mean±SEM. **p

    Techniques Used: Staining

    29) Product Images from "Golgi Tubule Traffic and the Effects of Brefeldin A Visualized in Living Cells "

    Article Title: Golgi Tubule Traffic and the Effects of Brefeldin A Visualized in Living Cells

    Journal: The Journal of Cell Biology

    doi:

    Microtubule depolymerization delays onset but not the kinetics of Golgi blinkout in BFA-treated cells. GFP-GalTase– expressing cells were placed on ice for 20 min, and nocodazole (1 μg/ml) was added to depolymerize microtubules ( Cole et al., 1996 b ). Cells were warmed to 37°C in the presence of BFA. Cells were then imaged at 37°C using a cooled CCD microscope system. Images shown begin at 4 min ( 4:00 ) after warm-up and extend until forty min ( 40:00 ). Very little change in Golgi morphology at the light microscope level occurred during the first 30 min of BFA treatment, with no tubules observed. Beginning at 36 min 30 s ( 36:30 ), however, GFP-GalTase redistributed into the ER, spreading throughout this compartment within 50 s ( 37:20 ). Thus, microtubule disruption delays onset of Golgi blinkout but does not affect its rapid kinetics. Bar, 5 μm . See Quicktime movie sequence at http://dir.nichd.nih.gov/CBMB/pb4labob.htm .
    Figure Legend Snippet: Microtubule depolymerization delays onset but not the kinetics of Golgi blinkout in BFA-treated cells. GFP-GalTase– expressing cells were placed on ice for 20 min, and nocodazole (1 μg/ml) was added to depolymerize microtubules ( Cole et al., 1996 b ). Cells were warmed to 37°C in the presence of BFA. Cells were then imaged at 37°C using a cooled CCD microscope system. Images shown begin at 4 min ( 4:00 ) after warm-up and extend until forty min ( 40:00 ). Very little change in Golgi morphology at the light microscope level occurred during the first 30 min of BFA treatment, with no tubules observed. Beginning at 36 min 30 s ( 36:30 ), however, GFP-GalTase redistributed into the ER, spreading throughout this compartment within 50 s ( 37:20 ). Thus, microtubule disruption delays onset of Golgi blinkout but does not affect its rapid kinetics. Bar, 5 μm . See Quicktime movie sequence at http://dir.nichd.nih.gov/CBMB/pb4labob.htm .

    Techniques Used: Expressing, Microscopy, Light Microscopy, Sequencing

    30) Product Images from "Localization of Mad2 to Kinetochores Depends on Microtubule Attachment, Not Tension "

    Article Title: Localization of Mad2 to Kinetochores Depends on Microtubule Attachment, Not Tension

    Journal: The Journal of Cell Biology

    doi:

    Mad2 rebinds PtK 1 kinetochores after microtubule depolymerization. This metaphase cell was treated with 20 μg/ml nocodazole for 20 min at 37°C before being fixed and processed for immunofluorescence. Coverslips were scribed before drug treatment to aid in subsequent identification of cells. Digital DIC ( black and white ) and Mad2 ( pink/red ) images were pseudocolored and overlaid. The majority of the 22 kinetochores are visible in this optical section.
    Figure Legend Snippet: Mad2 rebinds PtK 1 kinetochores after microtubule depolymerization. This metaphase cell was treated with 20 μg/ml nocodazole for 20 min at 37°C before being fixed and processed for immunofluorescence. Coverslips were scribed before drug treatment to aid in subsequent identification of cells. Digital DIC ( black and white ) and Mad2 ( pink/red ) images were pseudocolored and overlaid. The majority of the 22 kinetochores are visible in this optical section.

    Techniques Used: Immunofluorescence

    31) Product Images from "A Pore-forming Toxin Interacts with a GPI-anchored Protein and Causes Vacuolation of the Endoplasmic Reticulum "

    Article Title: A Pore-forming Toxin Interacts with a GPI-anchored Protein and Causes Vacuolation of the Endoplasmic Reticulum

    Journal: The Journal of Cell Biology

    doi:

    Proaerolysin-induced vacuolation is inhibited upon depolymerization of the microtubule network by nocodazole. Cells were treated with ( b and d ) or without ( a and c ) 10 μM of nocodazole for 1 h at 37°C before the addition of the toxin. The microtubule-depolymerizing drug remained present during the toxin treatment. After 60 min, cells were fixed with methanol and stained with anti-calnexin antibodies ( a and b ) or anti-tubulin antibodies. Bar, 6.7 μm
    Figure Legend Snippet: Proaerolysin-induced vacuolation is inhibited upon depolymerization of the microtubule network by nocodazole. Cells were treated with ( b and d ) or without ( a and c ) 10 μM of nocodazole for 1 h at 37°C before the addition of the toxin. The microtubule-depolymerizing drug remained present during the toxin treatment. After 60 min, cells were fixed with methanol and stained with anti-calnexin antibodies ( a and b ) or anti-tubulin antibodies. Bar, 6.7 μm

    Techniques Used: Staining

    32) Product Images from "Coupling between apical tension and basal adhesion allow epithelia to collectively sense and respond to substrate topography over long distances"

    Article Title: Coupling between apical tension and basal adhesion allow epithelia to collectively sense and respond to substrate topography over long distances

    Journal: Integrative biology : quantitative biosciences from nano to macro

    doi: 10.1039/c5ib00240k

    Treatment of cells with small-molecule drugs that modulate cell physicochemical properties affects their response to topography. MDCK cells treated with Y-27632, blebbistatin, or ML-7 do not clear, while untreated cells and those treated with Nocodazole
    Figure Legend Snippet: Treatment of cells with small-molecule drugs that modulate cell physicochemical properties affects their response to topography. MDCK cells treated with Y-27632, blebbistatin, or ML-7 do not clear, while untreated cells and those treated with Nocodazole

    Techniques Used:

    33) Product Images from "Human Papillomavirus Episome Stability Is Reduced by Aphidicolin and Controlled by DNA Damage Response Pathways"

    Article Title: Human Papillomavirus Episome Stability Is Reduced by Aphidicolin and Controlled by DNA Damage Response Pathways

    Journal: Journal of Virology

    doi: 10.1128/JVI.03473-12

    FACS analysis reveals effects of inhibitors used to activate cell cycle check points. (A) Effects of increasing doses of aphidicolin on arresting cells in the S-phase and at the G 1 /S boundary. (B) Nocodazole causes accumulation of cells in the G 2 /M phase.
    Figure Legend Snippet: FACS analysis reveals effects of inhibitors used to activate cell cycle check points. (A) Effects of increasing doses of aphidicolin on arresting cells in the S-phase and at the G 1 /S boundary. (B) Nocodazole causes accumulation of cells in the G 2 /M phase.

    Techniques Used: FACS

    Inhibitors of cell cycle progression were tested for effects on the antiviral activity of compound PA25. (A) W12E cells were treated with 250 nM nocodazole (Noc; Sigma catalog no. M1404) with or without 1 μM PA25 ( n = 6). (B) W12E cells were treated
    Figure Legend Snippet: Inhibitors of cell cycle progression were tested for effects on the antiviral activity of compound PA25. (A) W12E cells were treated with 250 nM nocodazole (Noc; Sigma catalog no. M1404) with or without 1 μM PA25 ( n = 6). (B) W12E cells were treated

    Techniques Used: Activity Assay

    34) Product Images from "Intracellular ion and protein nanoparticle-induced osmotic pressure modify astrocyte swelling and brain edema in response to glutamate stimuli"

    Article Title: Intracellular ion and protein nanoparticle-induced osmotic pressure modify astrocyte swelling and brain edema in response to glutamate stimuli

    Journal: Redox Biology

    doi: 10.1016/j.redox.2019.101112

    GFAP tension changes in U87 cells produced by suffering hypotonic pressure and the mechanism of action. (A) Representative images of normalized CFP/FRET ratios of GFAP tension subjected to isotonic treatment alone (row 1), with cytochalasin D (row 2,10 μM), nocodazole (row 3, 100 μM), or both agents (row 4). Cell volumes were determined at the initial and final times (right panel). (C) MF and MT tension after being subjected to isotonic treatment alone (row 1 and 3), MF with nocodazole (row 2) and MT with cytochalasin D (row 4). (F) GFAP tension in response to hypotonic stimuli alone (row 1), with 2,3-Butanedione monoxime (row 2, 10 mM), ciliobrevin D (row 3, 20 μM), SB-715992 (row 4, 1 μM), or both agents (row 5). (H) MF and MT tension in response to hypertonic stimulation alone (row 1 and 3), MF with 2,3-Butanedione monoxime (row 2), MT with Cilio D (row 4) or MT with SB-715992 (row 5). (B, D, G, and I) Mean values of normalized CFP/FRET ratios in GFAP (2 A and 2 F), MF and MT (2 C and 2 H) under isotonic or hypo-osmotic pressure responses to different reagents. The dark-blue calibration bar indicates the smallest tension (0.10), whereas red indicates the largest tension (2.85). (E) The osmotic pressure of cytoplasm in U87 cells in response to isosmotic pressure and with cytochalasin D, nocodazole, or both agents. Average of ≥ 5 experiments ± SEM. * **p
    Figure Legend Snippet: GFAP tension changes in U87 cells produced by suffering hypotonic pressure and the mechanism of action. (A) Representative images of normalized CFP/FRET ratios of GFAP tension subjected to isotonic treatment alone (row 1), with cytochalasin D (row 2,10 μM), nocodazole (row 3, 100 μM), or both agents (row 4). Cell volumes were determined at the initial and final times (right panel). (C) MF and MT tension after being subjected to isotonic treatment alone (row 1 and 3), MF with nocodazole (row 2) and MT with cytochalasin D (row 4). (F) GFAP tension in response to hypotonic stimuli alone (row 1), with 2,3-Butanedione monoxime (row 2, 10 mM), ciliobrevin D (row 3, 20 μM), SB-715992 (row 4, 1 μM), or both agents (row 5). (H) MF and MT tension in response to hypertonic stimulation alone (row 1 and 3), MF with 2,3-Butanedione monoxime (row 2), MT with Cilio D (row 4) or MT with SB-715992 (row 5). (B, D, G, and I) Mean values of normalized CFP/FRET ratios in GFAP (2 A and 2 F), MF and MT (2 C and 2 H) under isotonic or hypo-osmotic pressure responses to different reagents. The dark-blue calibration bar indicates the smallest tension (0.10), whereas red indicates the largest tension (2.85). (E) The osmotic pressure of cytoplasm in U87 cells in response to isosmotic pressure and with cytochalasin D, nocodazole, or both agents. Average of ≥ 5 experiments ± SEM. * **p

    Techniques Used: Produced

    35) Product Images from "Hyperphosphorylated tau causes reduced hippocampal CA1 excitability by relocating the axon initial segment"

    Article Title: Hyperphosphorylated tau causes reduced hippocampal CA1 excitability by relocating the axon initial segment

    Journal: Acta Neuropathologica

    doi: 10.1007/s00401-017-1674-1

    Pseudo-phosphorylated tau relocates the AIS down the axon in a process mediated by microtubules. Representative photomicrograph of A14- ( top ) and E14-tau-EGFP ( lower ) transfected hippocampal neurons stained for ankyrin G a 24 h and b 48 h after transfection. Arrows indicate the start, middle and end of the AIS as determined by quantitative analysis of the c , d axonal fluorescence profile. The dashed line indicates the normalized detection threshold. Quantification of ankyrin G labeling in neurons transfected with E14- ( blue ) and A14-tau ( black ) for e 24 h (start p = 0.48, middle p = 0.94, end p = 0.54; E14 n = 61, A14 n = 46) and f 48 h (start p = 0.0001, middle p = 0.0027, end p = 0.0017; E14 n = 47, A14 n = 62). AIS location of E14- and A14-tau-transfected neurons labeled for g βIV spectrin (start p = 0.0041, middle p = 0.0024, end p = 0.0084; E14 n = 55, A14 n = 52) and h Na V 1.6 (start p = 0.0042, middle p = 0.0045, end p = 0.0316; E14 n = 53, A14 n = 53). Using ankyrin G staining, the role of site-specific phosphorylation in AIS relocation was determined: i AT180 ( start p = 0.0138, middle p = 0.44, end p = 0.76; AT180E n = 53, AT180A n = 52), j 12E8 (start p = 0.0183, middle p = 0.0352, end p = 0.09; 12E8E n = 54, 12E8A n = 51), and k PFH1 (start p = 0.34, middle p = 0.56, end p = 0.75; PHF1E n = 50, PHF1A n = 38). l Treatment with 0.1 µM taxol ( magenta ) prevents AIS relocation (start, ANOVA, F = 8.05, p = 0.0005; middle, ANOVA, F = 4.057, p = 0.0191; end, ANOVA, F = 6.614, p = 0.0017; E14 n = 57, A14 n = 54, E14+ taxol n = 51). m Microtubule destabilization with 0.05 µM nocodazole ( green ) relocates the AIS ( gray , control, start: p = 0.0169, middle: p = 0.0297, end: p = 0.0856; nocodazole n = 54, control n = 55). * p
    Figure Legend Snippet: Pseudo-phosphorylated tau relocates the AIS down the axon in a process mediated by microtubules. Representative photomicrograph of A14- ( top ) and E14-tau-EGFP ( lower ) transfected hippocampal neurons stained for ankyrin G a 24 h and b 48 h after transfection. Arrows indicate the start, middle and end of the AIS as determined by quantitative analysis of the c , d axonal fluorescence profile. The dashed line indicates the normalized detection threshold. Quantification of ankyrin G labeling in neurons transfected with E14- ( blue ) and A14-tau ( black ) for e 24 h (start p = 0.48, middle p = 0.94, end p = 0.54; E14 n = 61, A14 n = 46) and f 48 h (start p = 0.0001, middle p = 0.0027, end p = 0.0017; E14 n = 47, A14 n = 62). AIS location of E14- and A14-tau-transfected neurons labeled for g βIV spectrin (start p = 0.0041, middle p = 0.0024, end p = 0.0084; E14 n = 55, A14 n = 52) and h Na V 1.6 (start p = 0.0042, middle p = 0.0045, end p = 0.0316; E14 n = 53, A14 n = 53). Using ankyrin G staining, the role of site-specific phosphorylation in AIS relocation was determined: i AT180 ( start p = 0.0138, middle p = 0.44, end p = 0.76; AT180E n = 53, AT180A n = 52), j 12E8 (start p = 0.0183, middle p = 0.0352, end p = 0.09; 12E8E n = 54, 12E8A n = 51), and k PFH1 (start p = 0.34, middle p = 0.56, end p = 0.75; PHF1E n = 50, PHF1A n = 38). l Treatment with 0.1 µM taxol ( magenta ) prevents AIS relocation (start, ANOVA, F = 8.05, p = 0.0005; middle, ANOVA, F = 4.057, p = 0.0191; end, ANOVA, F = 6.614, p = 0.0017; E14 n = 57, A14 n = 54, E14+ taxol n = 51). m Microtubule destabilization with 0.05 µM nocodazole ( green ) relocates the AIS ( gray , control, start: p = 0.0169, middle: p = 0.0297, end: p = 0.0856; nocodazole n = 54, control n = 55). * p

    Techniques Used: Transfection, Staining, Fluorescence, Labeling

    36) Product Images from "Hypoxia and cell cycle regulation of the von Hippel-Lindau tumor suppressor"

    Article Title: Hypoxia and cell cycle regulation of the von Hippel-Lindau tumor suppressor

    Journal: Oncogene

    doi: 10.1038/onc.2010.395

    pVHL levels fluctuate through the cell cycle Cell cycle synchronization of HeLa and 786-O G7F RCC cells was performed using double thymidine block or thymidine plus nocodazole block as described in the Materials and Methods. Cells were released from synchronization and harvested at the indicated times. (a) Expression of the indicated proteins in HeLa cells or 786-O G7F RCC cells was examined by western blotting, and β-actin detection was used to demonstrate equal loading. (b) Analyses of HeLa cells were performed to confirm cell cycle arrest and release using propidium iodide staining and flow cytometry. Histograms are shown as well as the corresponding numbers of cells in G1, S, and the G2/M phases. The 0 time point indicates cells arrested with the indicated agents but not released from block. A representative experiment of three performed is shown. Similar results were obtained using 786-O G7F RCC cells (data not shown). (c) HeLa cells were synchronized by thymidine plus nocodazole block. Three hrs after release pulse-chase studies were performed as described in Figure 2 and the Materials and Methods.
    Figure Legend Snippet: pVHL levels fluctuate through the cell cycle Cell cycle synchronization of HeLa and 786-O G7F RCC cells was performed using double thymidine block or thymidine plus nocodazole block as described in the Materials and Methods. Cells were released from synchronization and harvested at the indicated times. (a) Expression of the indicated proteins in HeLa cells or 786-O G7F RCC cells was examined by western blotting, and β-actin detection was used to demonstrate equal loading. (b) Analyses of HeLa cells were performed to confirm cell cycle arrest and release using propidium iodide staining and flow cytometry. Histograms are shown as well as the corresponding numbers of cells in G1, S, and the G2/M phases. The 0 time point indicates cells arrested with the indicated agents but not released from block. A representative experiment of three performed is shown. Similar results were obtained using 786-O G7F RCC cells (data not shown). (c) HeLa cells were synchronized by thymidine plus nocodazole block. Three hrs after release pulse-chase studies were performed as described in Figure 2 and the Materials and Methods.

    Techniques Used: Blocking Assay, Expressing, Western Blot, Staining, Flow Cytometry, Cytometry, Pulse Chase

    37) Product Images from "Adenovirus E4orf4 Protein-Induced Death of p53−/− H1299 Human Cancer Cells Follows a G1 Arrest of both Tetraploid and Diploid Cells due to a Failure To Initiate DNA Synthesis"

    Article Title: Adenovirus E4orf4 Protein-Induced Death of p53−/− H1299 Human Cancer Cells Follows a G1 Arrest of both Tetraploid and Diploid Cells due to a Failure To Initiate DNA Synthesis

    Journal: Journal of Virology

    doi: 10.1128/JVI.01242-13

    Analysis of mitotic cells and markers. (A) Detection of cyclins B1 and E in mitotic and E4orf4-expressing cells. H1299 cells were infected with AdrtTA or AdE4orf4, or they were treated with nocodazole, and at 1 or 3 days postinfection, cells were separated
    Figure Legend Snippet: Analysis of mitotic cells and markers. (A) Detection of cyclins B1 and E in mitotic and E4orf4-expressing cells. H1299 cells were infected with AdrtTA or AdE4orf4, or they were treated with nocodazole, and at 1 or 3 days postinfection, cells were separated

    Techniques Used: Expressing, Infection

    38) Product Images from "Non-catalytic participation of the Pin1 peptidyl-prolyl isomerase domain in target binding"

    Article Title: Non-catalytic participation of the Pin1 peptidyl-prolyl isomerase domain in target binding

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2013.00018

    GST pull-downs with Pin1 mutants reveal two different types of binding proteins. (A) Large-scale GST pull-downs. One to two milligrams of nocodazole-treated HeLa lysates were incubated with GST fusion proteins bound to glutathione beads. Interacting proteins were run on a 5–12% gradient gel and stained with Coomassie Blue. (B) GST pull-downs were performed as in (A) with 1 mg of HeLa lysate. Interacting proteins were run on 10% SDS-PAGE, transferred to PVDF and blotted with the indicated antibody. To better compare the amount of fusion protein on the beads, 1/200 of each pull-down was run on a gel and stained with Coomassie Blue (PD). (C) Quantification of blots shown in (B) . Results are the mean of three independent pull-down experiments ±S.E.M.
    Figure Legend Snippet: GST pull-downs with Pin1 mutants reveal two different types of binding proteins. (A) Large-scale GST pull-downs. One to two milligrams of nocodazole-treated HeLa lysates were incubated with GST fusion proteins bound to glutathione beads. Interacting proteins were run on a 5–12% gradient gel and stained with Coomassie Blue. (B) GST pull-downs were performed as in (A) with 1 mg of HeLa lysate. Interacting proteins were run on 10% SDS-PAGE, transferred to PVDF and blotted with the indicated antibody. To better compare the amount of fusion protein on the beads, 1/200 of each pull-down was run on a gel and stained with Coomassie Blue (PD). (C) Quantification of blots shown in (B) . Results are the mean of three independent pull-down experiments ±S.E.M.

    Techniques Used: Binding Assay, Incubation, Staining, SDS Page

    p54nrb/NonO binding to R68A/R69A is not due to protein abundance. (A) One to two milligrams of nocodazole-treated HeLa lysates were incubated with GST fusion proteins bound to glutathione beads. Serial dilutions of the pull-down were run on a 5–12% gradient gel, and immunoblotted with anti-p54nrb/NonO. (B) Hundred micrograms of nocodazole-treated HeLa lysates (N/C) were incubated with GST fusion proteins bound to glutathione beads. Interacting proteins were run on a 5–12% gradient gel and stained with Coomassie Blue. (C) HeLa cells were either transfected with HA-NonO or left untransfected (NT). Lysates were used in GST pull-downs (PD's) as above and immunoblotted with the indicated antibody. Endogenous NonO is indicated with End. (D) HeLa cells were transfected with HA-NonO or HA-Cdc25C or left untransfected (NT). Lysates were used in pull-downs (PD) as above and transfected proteins were detected with anti-HA. For the combined sample, 1 mg of HA-NonO lysate was mixed with 1 mg of HA-Cdc25C lysate.
    Figure Legend Snippet: p54nrb/NonO binding to R68A/R69A is not due to protein abundance. (A) One to two milligrams of nocodazole-treated HeLa lysates were incubated with GST fusion proteins bound to glutathione beads. Serial dilutions of the pull-down were run on a 5–12% gradient gel, and immunoblotted with anti-p54nrb/NonO. (B) Hundred micrograms of nocodazole-treated HeLa lysates (N/C) were incubated with GST fusion proteins bound to glutathione beads. Interacting proteins were run on a 5–12% gradient gel and stained with Coomassie Blue. (C) HeLa cells were either transfected with HA-NonO or left untransfected (NT). Lysates were used in GST pull-downs (PD's) as above and immunoblotted with the indicated antibody. Endogenous NonO is indicated with End. (D) HeLa cells were transfected with HA-NonO or HA-Cdc25C or left untransfected (NT). Lysates were used in pull-downs (PD) as above and transfected proteins were detected with anti-HA. For the combined sample, 1 mg of HA-NonO lysate was mixed with 1 mg of HA-Cdc25C lysate.

    Techniques Used: Binding Assay, Incubation, Staining, Transfection

    39) Product Images from "The spindle checkpoint requires cyclin-dependent kinase activity"

    Article Title: The spindle checkpoint requires cyclin-dependent kinase activity

    Journal: Genes & Development

    doi: 10.1101/gad.267603

    Cdk1 inhibition affects Cdc20 phosphorylation and binding to Cdc27 and Mad2 in Hela cells. Cdc20 was immunoprecipitated (Ip) and associated Cdc27 or Mad2 were detected by immunoblot (Ib) from checkpoint-arrested cells taken at the indicated time points from Roscovitine addition ( A ) and nocodazole washout ( B ). Optical density values of Cdc27 bound to Cdc20 are indicated (Cdc27 od; arbitrary units). (Mk Ip) Mock precipitations. ( C, left ) Immunoblot of Cdc20 from thymidine-arrested (T) and from nocodazole-arrested HeLa cells taken at the indicated time points after Roscovitine addition. ( Right ) Immunoblot of Cdc20 from thymidine-arrested cells (T), from nocodazole-arrested cells nontreated (0) and treated (λPP) in vitro with λ protein phosphatase and from nocodazole-arrested cells after 20 min from Roscovitine addition (20).
    Figure Legend Snippet: Cdk1 inhibition affects Cdc20 phosphorylation and binding to Cdc27 and Mad2 in Hela cells. Cdc20 was immunoprecipitated (Ip) and associated Cdc27 or Mad2 were detected by immunoblot (Ib) from checkpoint-arrested cells taken at the indicated time points from Roscovitine addition ( A ) and nocodazole washout ( B ). Optical density values of Cdc27 bound to Cdc20 are indicated (Cdc27 od; arbitrary units). (Mk Ip) Mock precipitations. ( C, left ) Immunoblot of Cdc20 from thymidine-arrested (T) and from nocodazole-arrested HeLa cells taken at the indicated time points after Roscovitine addition. ( Right ) Immunoblot of Cdc20 from thymidine-arrested cells (T), from nocodazole-arrested cells nontreated (0) and treated (λPP) in vitro with λ protein phosphatase and from nocodazole-arrested cells after 20 min from Roscovitine addition (20).

    Techniques Used: Inhibition, Binding Assay, Immunoprecipitation, In Vitro

    Cdk inhibition overrides checkpoint-dependent arrest in HeLa cells. Checkpoint-arrested HeLa cells were collected 14 h after nocodazole addition. DMSO, as control, or Roscovitine were then added in the continuous presence of nocodazole and samples taken at the indicated time points. ( A ) Chromosomes were stained with Giemsa. Arrowheads (Control) or brackets (Roscovitine) indicate maintenance or loss of sister chromatid cohesion. ( B ) Securin and cyclin B1 were visualized by immunoblot from total cell lysates; securin and cyclin B1 were also visualized from cells taken after nocodazole washout (NWO). In A , sister chromatid cohesion was lost in about 80% of cells 20 min after Roscovitine addition. ( C ) Cdc20 or Cdc27 were immunoprecipitated from samples taken at the indicated time points from Roscovitine addition (Ip) and associated Cdc27, Cdc20, and Mad2 were detected by immunoblot (Ib); the total amount of Mad2 is also shown. (Mk Ip) Mock precipitations; (Ig) immunoglobulin. ( D ) Checkpoint-arrested HeLa cells were collected 14 h after nocodazole addition and split into seven dishes. Then, DMSO (lane 1 ), Alsterpaullone (800 nM; lane 2 ), Indirubin 3′ monoxime (6 μM; lane 3 ), Purvalanol A (600 nM; lane 4 ), UO 126 (30 μM; lane 5 ), SB 203589 (30 μM; lane 6 ), and H89 (30 μM; lane 7 ), were individually added in the continuous presence of nocodazole, and cells harvested after additional 40 min incubation. Cdc20 was immunoprecipitated (Ip) and associated Cdc27 and Mad2 were detected by immunoblot (Ib), the total amount of Mad2, cyclin B1, and cdk1 is also shown. UO 126, SB 203589, and H89 were also tested in a range of concentration from 30 nM to 50 μM giving similar results (not shown).
    Figure Legend Snippet: Cdk inhibition overrides checkpoint-dependent arrest in HeLa cells. Checkpoint-arrested HeLa cells were collected 14 h after nocodazole addition. DMSO, as control, or Roscovitine were then added in the continuous presence of nocodazole and samples taken at the indicated time points. ( A ) Chromosomes were stained with Giemsa. Arrowheads (Control) or brackets (Roscovitine) indicate maintenance or loss of sister chromatid cohesion. ( B ) Securin and cyclin B1 were visualized by immunoblot from total cell lysates; securin and cyclin B1 were also visualized from cells taken after nocodazole washout (NWO). In A , sister chromatid cohesion was lost in about 80% of cells 20 min after Roscovitine addition. ( C ) Cdc20 or Cdc27 were immunoprecipitated from samples taken at the indicated time points from Roscovitine addition (Ip) and associated Cdc27, Cdc20, and Mad2 were detected by immunoblot (Ib); the total amount of Mad2 is also shown. (Mk Ip) Mock precipitations; (Ig) immunoglobulin. ( D ) Checkpoint-arrested HeLa cells were collected 14 h after nocodazole addition and split into seven dishes. Then, DMSO (lane 1 ), Alsterpaullone (800 nM; lane 2 ), Indirubin 3′ monoxime (6 μM; lane 3 ), Purvalanol A (600 nM; lane 4 ), UO 126 (30 μM; lane 5 ), SB 203589 (30 μM; lane 6 ), and H89 (30 μM; lane 7 ), were individually added in the continuous presence of nocodazole, and cells harvested after additional 40 min incubation. Cdc20 was immunoprecipitated (Ip) and associated Cdc27 and Mad2 were detected by immunoblot (Ib), the total amount of Mad2, cyclin B1, and cdk1 is also shown. UO 126, SB 203589, and H89 were also tested in a range of concentration from 30 nM to 50 μM giving similar results (not shown).

    Techniques Used: Inhibition, Staining, Immunoprecipitation, Incubation, Concentration Assay

    Cdk activities in checkpoint-arrested egg extracts. MPF (□; cyclin B–cdk1) and cdk2 (▪) kinase activities and autoradiographs of [ 35 S]-labeled extracts proteins (the position of cyclin B is indicated) from portions of a cycling Xenopus egg extract during incubation at 23°C. ( A ) Control extract (+nocodazole). ( B ) Checkpoint-arrested extract (+demembranated sperm nuclei + nocodazole). Portions of the checkpoint-arrested extract received after 22 min of incubation: ( C ) GST–Cip1 (100 nM). ( D ) Roscovitine (2 μM; in DMSO). ( E ) MAPK activity in a checkpoint-arrested extract. ( F ) MAPK activity in a portion of the checkpoint-arrested extract that received GST–Cip1 (100 nM) after 22 min of incubation. The addition of GST or DMSO to checkpoint-arrested extracts did not reverse cell cycle arrest (data not shown).
    Figure Legend Snippet: Cdk activities in checkpoint-arrested egg extracts. MPF (□; cyclin B–cdk1) and cdk2 (▪) kinase activities and autoradiographs of [ 35 S]-labeled extracts proteins (the position of cyclin B is indicated) from portions of a cycling Xenopus egg extract during incubation at 23°C. ( A ) Control extract (+nocodazole). ( B ) Checkpoint-arrested extract (+demembranated sperm nuclei + nocodazole). Portions of the checkpoint-arrested extract received after 22 min of incubation: ( C ) GST–Cip1 (100 nM). ( D ) Roscovitine (2 μM; in DMSO). ( E ) MAPK activity in a checkpoint-arrested extract. ( F ) MAPK activity in a portion of the checkpoint-arrested extract that received GST–Cip1 (100 nM) after 22 min of incubation. The addition of GST or DMSO to checkpoint-arrested extracts did not reverse cell cycle arrest (data not shown).

    Techniques Used: Labeling, Incubation, Activity Assay

    40) Product Images from "Identification of an Overlapping Binding Domain on Cdc20 for Mad2 and Anaphase-Promoting Complex: Model for Spindle Checkpoint Regulation"

    Article Title: Identification of an Overlapping Binding Domain on Cdc20 for Mad2 and Anaphase-Promoting Complex: Model for Spindle Checkpoint Regulation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.15.5190-5199.2001

    Phenotype of 293T cells overexpressing WT Cdc20 and mutants. (A) 293T cells were transfected with pFlag-CMV vectors encoding WT Cdc20, 1–153 Cdc20, 1–101 Cdc20, or 211–499 Cdc20. After transfection for 48 h, the transfected cells were detected by immunofluorescence using anti-Flag M5 antibody. DNA was stained with propidium iodide. (B) The above transfected 293T cells were further treated with nocodazole for 18 h. The mitotic index was analyzed by fluorescence microscopy using anti-Flag M5 antibody for positive cells and propidium iodide for chromosome DNA. The shaded bars and error bars represent the means and standard deviations, respectively, from at least two independent assessments of 100 cells each in a single experiment; similar results were obtained in two independent experiments. (C) 293T cells were transfected with pFlag-CMV vectors encoding 1–153 Cdc20, 1–101 Cdc20, or vector control for 48 h and were further treated with nocodazole for 18 h. The levels of the corresponding truncated mutant protein, endogenous Cdc20, and Mad2 were detected by Western blotting with anti-Flag M5 antibody, goat anti-Cdc20 polyclonal antibody, and mouse anti-Mad2 monoclonal antibody. Cell lysates were immunoprecipitated with goat anti-Cdc20 polyclonal antibody. Precipitated proteins were separated by SDS-PAGE, transferred onto nylon membranes, and Western blotted with mouse anti-Mad2 monoclonal antibody. PI, propidium iodide; Noc, nocodazole; WB, Western blotting; IP, immunoprecipitation.
    Figure Legend Snippet: Phenotype of 293T cells overexpressing WT Cdc20 and mutants. (A) 293T cells were transfected with pFlag-CMV vectors encoding WT Cdc20, 1–153 Cdc20, 1–101 Cdc20, or 211–499 Cdc20. After transfection for 48 h, the transfected cells were detected by immunofluorescence using anti-Flag M5 antibody. DNA was stained with propidium iodide. (B) The above transfected 293T cells were further treated with nocodazole for 18 h. The mitotic index was analyzed by fluorescence microscopy using anti-Flag M5 antibody for positive cells and propidium iodide for chromosome DNA. The shaded bars and error bars represent the means and standard deviations, respectively, from at least two independent assessments of 100 cells each in a single experiment; similar results were obtained in two independent experiments. (C) 293T cells were transfected with pFlag-CMV vectors encoding 1–153 Cdc20, 1–101 Cdc20, or vector control for 48 h and were further treated with nocodazole for 18 h. The levels of the corresponding truncated mutant protein, endogenous Cdc20, and Mad2 were detected by Western blotting with anti-Flag M5 antibody, goat anti-Cdc20 polyclonal antibody, and mouse anti-Mad2 monoclonal antibody. Cell lysates were immunoprecipitated with goat anti-Cdc20 polyclonal antibody. Precipitated proteins were separated by SDS-PAGE, transferred onto nylon membranes, and Western blotted with mouse anti-Mad2 monoclonal antibody. PI, propidium iodide; Noc, nocodazole; WB, Western blotting; IP, immunoprecipitation.

    Techniques Used: Transfection, Immunofluorescence, Staining, Fluorescence, Microscopy, Plasmid Preparation, Mutagenesis, Western Blot, Immunoprecipitation, SDS Page

    Mitotic checkpoint-regulated association of Cdc20 with Mad2 and APC. (A) A549 cells were synchronized at prometaphase by nocodazole block. Mitotically arrested cells were collected by shake-off. Cells released into fresh medium were collected at indicated time points. To arrest cells in mitosis without activating the checkpoint, A549 cells were released from nocodazole into fresh medium containing 10 μM MG-132 (lane 10). Cell extracts (500 μg) were prepared at indicated time points and were immunoprecipitated with goat anti-Cdc20 polyclonal antibody, and the immunoprecipitates were analyzed by Western blotting with mouse anti-Mad2 monoclonal antibody (upper panel) and rabbit anti-APC2 polyclonal antibody (lower panel). Asynchronous cells (lane 1) and paclitaxel (lane 7) were included as controls. (B) The levels of Cdc20, Mad2, APC2, and cyclin B proteins at indicated time points and conditions were determined by Western blot analysis. (C) 293T cells were transfected with mock vector or Flag-tagged Cdc20 for 24 h. Transfected cells were treated with (+) or without (−) 0.4 μg of nocodazole/ml for 18 h. Cell extracts (1 mg) were immunoprecipitated with anti-Flag M2-conjugated agarose beads, and the immunoprecipitates were subjected to Western blot analysis with goat anti-Mad2 polyclonal antibody (upper panel). The level of Flag-tagged Cdc20 was determined by Western blotting with anti-Flag M5 monoclonal antibody (lower panel). (D) A549 cells were arrested in mitosis by nocodazole block, and cell extracts were immunoprecipitated with mouse anti-Mad2 monoclonal antibody either before (lane 1) or after (lane 2) immunodepletion with goat anti-Cdc20 polyclonal antibody. The immunoprecipitates were analyzed by Western blotting with rabbit anti-APC2 polyclonal antibody (upper panel). The amounts of Cdc20, APC2, Mad2, and p21 proteins in the supernatant before and after immunodepletion of Cdc20 were determined by Western blotting as indicated. IP, immunoprecipitation; NS, nonsynchronous; Noc, nocodazole; WB, Western blotting; A, asynchronous.
    Figure Legend Snippet: Mitotic checkpoint-regulated association of Cdc20 with Mad2 and APC. (A) A549 cells were synchronized at prometaphase by nocodazole block. Mitotically arrested cells were collected by shake-off. Cells released into fresh medium were collected at indicated time points. To arrest cells in mitosis without activating the checkpoint, A549 cells were released from nocodazole into fresh medium containing 10 μM MG-132 (lane 10). Cell extracts (500 μg) were prepared at indicated time points and were immunoprecipitated with goat anti-Cdc20 polyclonal antibody, and the immunoprecipitates were analyzed by Western blotting with mouse anti-Mad2 monoclonal antibody (upper panel) and rabbit anti-APC2 polyclonal antibody (lower panel). Asynchronous cells (lane 1) and paclitaxel (lane 7) were included as controls. (B) The levels of Cdc20, Mad2, APC2, and cyclin B proteins at indicated time points and conditions were determined by Western blot analysis. (C) 293T cells were transfected with mock vector or Flag-tagged Cdc20 for 24 h. Transfected cells were treated with (+) or without (−) 0.4 μg of nocodazole/ml for 18 h. Cell extracts (1 mg) were immunoprecipitated with anti-Flag M2-conjugated agarose beads, and the immunoprecipitates were subjected to Western blot analysis with goat anti-Mad2 polyclonal antibody (upper panel). The level of Flag-tagged Cdc20 was determined by Western blotting with anti-Flag M5 monoclonal antibody (lower panel). (D) A549 cells were arrested in mitosis by nocodazole block, and cell extracts were immunoprecipitated with mouse anti-Mad2 monoclonal antibody either before (lane 1) or after (lane 2) immunodepletion with goat anti-Cdc20 polyclonal antibody. The immunoprecipitates were analyzed by Western blotting with rabbit anti-APC2 polyclonal antibody (upper panel). The amounts of Cdc20, APC2, Mad2, and p21 proteins in the supernatant before and after immunodepletion of Cdc20 were determined by Western blotting as indicated. IP, immunoprecipitation; NS, nonsynchronous; Noc, nocodazole; WB, Western blotting; A, asynchronous.

    Techniques Used: Blocking Assay, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation

    A549 cells were transfected with pEGFP vector encoding WT Cdc20 or 1–153 Cdc20 mutant for 24 h and were further treated with nocodazole (A) or paclitaxel (B) for 18 h. GFP fusion constructs were directly visualized by autofluorescence. Multinucleated cells with aberrantly shaped nuclei and apoptotic cells with nuclear condensation and fragmentation were determined by counting 100 GFP-positive cells visualized with propidium iodide staining. The data show the averages and standard deviations derived from three experiments. (C) Representative morphology of paclitaxel-treated A549 cells transfected with 1–153 Cdc20.
    Figure Legend Snippet: A549 cells were transfected with pEGFP vector encoding WT Cdc20 or 1–153 Cdc20 mutant for 24 h and were further treated with nocodazole (A) or paclitaxel (B) for 18 h. GFP fusion constructs were directly visualized by autofluorescence. Multinucleated cells with aberrantly shaped nuclei and apoptotic cells with nuclear condensation and fragmentation were determined by counting 100 GFP-positive cells visualized with propidium iodide staining. The data show the averages and standard deviations derived from three experiments. (C) Representative morphology of paclitaxel-treated A549 cells transfected with 1–153 Cdc20.

    Techniques Used: Transfection, Plasmid Preparation, Mutagenesis, Construct, Staining, Derivative Assay

    Constitutive binding of 1–153 Cdc20 mutant to Mad2. 293T cells were transfected with equal amounts of indicated plasmids. After transfection for 24 h, cells were further treated with or without nocodazole for 18 h. Cells were harvested, and cell lysates were immunoprecipitated with anti-Flag agarose beads. Precipitated proteins were separated by SDS-PAGE, transferred onto nylon membranes, and Western blotted with goat anti-Mad2 polyclonal antibody (lower panel). The corresponding expression levels of WT and mutant proteins in transfected 293T cells were detected by Western blotting with anti-Flag M5 antibody (upper panel). Noc, nocodazole; IP, immunoprecipitation; WB, Western blotting.
    Figure Legend Snippet: Constitutive binding of 1–153 Cdc20 mutant to Mad2. 293T cells were transfected with equal amounts of indicated plasmids. After transfection for 24 h, cells were further treated with or without nocodazole for 18 h. Cells were harvested, and cell lysates were immunoprecipitated with anti-Flag agarose beads. Precipitated proteins were separated by SDS-PAGE, transferred onto nylon membranes, and Western blotted with goat anti-Mad2 polyclonal antibody (lower panel). The corresponding expression levels of WT and mutant proteins in transfected 293T cells were detected by Western blotting with anti-Flag M5 antibody (upper panel). Noc, nocodazole; IP, immunoprecipitation; WB, Western blotting.

    Techniques Used: Binding Assay, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Western Blot, Expressing

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    Article Snippet: .. Nocodazole (Sigma-Aldrich Co., St. Louis, MO, USA), MG132 (Wako Pure Chemicals Industries, Ltd, Osaka, Japan), ciliobrevin D (Merck KGaA, Darmstadt, Germany), and tubacin (Santa Cruz Biotechnologies, Inc., Dallas, TX, USA) were purchased commercially, solubilized in DMSO, and used at 10, 10, 20, and 10 μM, respectively, as working concentration. ..

    Incubation:

    Article Title: Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1
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    Article Title: Self-Assembly of the RZZ Complex into Filaments Drives Kinetochore Expansion in the Absence of Microtubule Attachment
    Article Snippet: .. To detach kinetochore crescents from centromeres, cells were incubated for 4 hr with 1 μM nocodazole followed by a 30-min incubation with nocodazole and 10 μM CDK1 inhibitor RO-3306 (Sigma-Aldrich). ..

    Article Title: ATP depletion during mitotic arrest induces mitotic slippage and APC/CCdh1-dependent cyclin B1 degradation
    Article Snippet: .. For synchronization at the G1/S border by double-thymidine block (DTB), cells grown on coverslips were incubated in growth medium containing 1 mM thymidine (Sigma, T9250) for 20 h. Cells were then released from the thymidine block by washing with thymidine-free medium (first release) and cultured in growth medium for 9 h. Subsequently, cells were subjected to the second thymidine block for an additional 16 h. For mitotic arrest, cells were synchronized at prometaphase with 100 ng/ml nocodazole (Sigma, M1404) or 1 μM Taxol for 16 h. Then, cells were treated with 6 mM 2-deoxyglucose (2-DG) (Sigma, D6134) and 10 mM sodium azide (NaN3 ) (Sigma, S2002). .. For inhibition of proteolysis, cells were treated with 10 μM MG132 (Sigma, C2211) 30 min before co-treatment with 6 mM 2-DG and 10 mM NaN3 .

    Blocking Assay:

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    Article Title: ATP depletion during mitotic arrest induces mitotic slippage and APC/CCdh1-dependent cyclin B1 degradation
    Article Snippet: .. For synchronization at the G1/S border by double-thymidine block (DTB), cells grown on coverslips were incubated in growth medium containing 1 mM thymidine (Sigma, T9250) for 20 h. Cells were then released from the thymidine block by washing with thymidine-free medium (first release) and cultured in growth medium for 9 h. Subsequently, cells were subjected to the second thymidine block for an additional 16 h. For mitotic arrest, cells were synchronized at prometaphase with 100 ng/ml nocodazole (Sigma, M1404) or 1 μM Taxol for 16 h. Then, cells were treated with 6 mM 2-deoxyglucose (2-DG) (Sigma, D6134) and 10 mM sodium azide (NaN3 ) (Sigma, S2002). .. For inhibition of proteolysis, cells were treated with 10 μM MG132 (Sigma, C2211) 30 min before co-treatment with 6 mM 2-DG and 10 mM NaN3 .

    Cell Culture:

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    Cell Cycle Assay:

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  • 99
    Millipore nocodazole
    (with 3 supplements). Delta does not decrease significantly in Taxol-treated IM cells. (A) Scatterplots of CenpA (green) - Hec1 (red) Delta versus interkinetochore (CenpA-CenpA) distance for untreated metaphase and Taxol-treated IM cells. PFA fixation, followed by methanol. Dashed lines denote mean. Mean ± standard deviation (SD) also shown numerically. ( B) IM cell with inner and outer kinetochore domains labeled via expression of CenpA-GFP (green) and immunostaining for Ndc80/Hec1 (red). Glutaraldehyde (GA) fixation. Notice that the intensity of staining is similar to Figure 1B although chromosome arms appear slightly more condensed. ( B’) Higher-magnification of sister kinetochores boxed in B. Scale bar = 500 nm. ( C) Scatterplots of CenpA-GFP (green) - Hec1 (red) Delta versus interkinetochore (CenpA-GFP – CenpA-GFP) distance for untreated, Taxol-, and <t>nocodazole-treated</t> IM cells. PFA fixation. Mean ± standard deviation (SD) shown. (D) As in C but cells are fixed with GA.
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    (with 3 supplements). Delta does not decrease significantly in Taxol-treated IM cells. (A) Scatterplots of CenpA (green) - Hec1 (red) Delta versus interkinetochore (CenpA-CenpA) distance for untreated metaphase and Taxol-treated IM cells. PFA fixation, followed by methanol. Dashed lines denote mean. Mean ± standard deviation (SD) also shown numerically. ( B) IM cell with inner and outer kinetochore domains labeled via expression of CenpA-GFP (green) and immunostaining for Ndc80/Hec1 (red). Glutaraldehyde (GA) fixation. Notice that the intensity of staining is similar to Figure 1B although chromosome arms appear slightly more condensed. ( B’) Higher-magnification of sister kinetochores boxed in B. Scale bar = 500 nm. ( C) Scatterplots of CenpA-GFP (green) - Hec1 (red) Delta versus interkinetochore (CenpA-GFP – CenpA-GFP) distance for untreated, Taxol-, and nocodazole-treated IM cells. PFA fixation. Mean ± standard deviation (SD) shown. (D) As in C but cells are fixed with GA.

    Journal: bioRxiv

    Article Title: kSHREC ‘Delta’ reflects the shape of kinetochore rather than intrakinetochore tension

    doi: 10.1101/811075

    Figure Lengend Snippet: (with 3 supplements). Delta does not decrease significantly in Taxol-treated IM cells. (A) Scatterplots of CenpA (green) - Hec1 (red) Delta versus interkinetochore (CenpA-CenpA) distance for untreated metaphase and Taxol-treated IM cells. PFA fixation, followed by methanol. Dashed lines denote mean. Mean ± standard deviation (SD) also shown numerically. ( B) IM cell with inner and outer kinetochore domains labeled via expression of CenpA-GFP (green) and immunostaining for Ndc80/Hec1 (red). Glutaraldehyde (GA) fixation. Notice that the intensity of staining is similar to Figure 1B although chromosome arms appear slightly more condensed. ( B’) Higher-magnification of sister kinetochores boxed in B. Scale bar = 500 nm. ( C) Scatterplots of CenpA-GFP (green) - Hec1 (red) Delta versus interkinetochore (CenpA-GFP – CenpA-GFP) distance for untreated, Taxol-, and nocodazole-treated IM cells. PFA fixation. Mean ± standard deviation (SD) shown. (D) As in C but cells are fixed with GA.

    Article Snippet: Taxol (Paclitaxel T7402-5MG; Sigma-Aldrich) and nocodazole (Calbiochem, #487928) were added ∼ 15 min prior to fixation to final concentrations of 10-μM and 3-μM respectively.

    Techniques: Standard Deviation, Labeling, Expressing, Immunostaining, Staining

    Kinetochore layers in IM cells are wide and their width changes upon Taxol and nocodazole treatments. (A) Examples of line-scans (white lines) across individual microtubules or kinetochore plates in cells fixed with Paraformaldehyde (PFA) or Glutaraldehyde (GA). ( B) Fluorescence profiles corresponding to line-scans shown in A. Markers are pixel intensities, lines are Gaussian fits. Values of the Full Width at Half-Maximum (FWHM) are shown for each profile. Notice that fluorescence peaks of inner- and outer-kinetochore layers are significantly wider than the peaks of individual microtubules. ( C) Box plots presenting measurements of FWHM for microtubules, inner- (CenpA-GFP), and outer- (Hec1) kinetochore layers in untreated, Taxol- and nocodazole-treated metaphases after PFA (left) and GA (right) fixation. Notice that FWHM of Hec1 layer increases in Taxol- and nocodazole-treated cells. Increase in CenpA-GFP layer is apparent after PFA but not after GA fixation. Student’s t-test p values are less than 10 −4 (****), 10 −5 (*****), or greater than 0.5 (NS).

    Journal: bioRxiv

    Article Title: kSHREC ‘Delta’ reflects the shape of kinetochore rather than intrakinetochore tension

    doi: 10.1101/811075

    Figure Lengend Snippet: Kinetochore layers in IM cells are wide and their width changes upon Taxol and nocodazole treatments. (A) Examples of line-scans (white lines) across individual microtubules or kinetochore plates in cells fixed with Paraformaldehyde (PFA) or Glutaraldehyde (GA). ( B) Fluorescence profiles corresponding to line-scans shown in A. Markers are pixel intensities, lines are Gaussian fits. Values of the Full Width at Half-Maximum (FWHM) are shown for each profile. Notice that fluorescence peaks of inner- and outer-kinetochore layers are significantly wider than the peaks of individual microtubules. ( C) Box plots presenting measurements of FWHM for microtubules, inner- (CenpA-GFP), and outer- (Hec1) kinetochore layers in untreated, Taxol- and nocodazole-treated metaphases after PFA (left) and GA (right) fixation. Notice that FWHM of Hec1 layer increases in Taxol- and nocodazole-treated cells. Increase in CenpA-GFP layer is apparent after PFA but not after GA fixation. Student’s t-test p values are less than 10 −4 (****), 10 −5 (*****), or greater than 0.5 (NS).

    Article Snippet: Taxol (Paclitaxel T7402-5MG; Sigma-Aldrich) and nocodazole (Calbiochem, #487928) were added ∼ 15 min prior to fixation to final concentrations of 10-μM and 3-μM respectively.

    Techniques: Fluorescence

    SAC maintenance and resolution in HeLa and hTERT-RPE1 cells treated with the phosphatase inhibitor Okadaic Acid Nocodazole-treated, prometaphase-arrested, ( A ) HeLa and ( B ) hTERT-RPE1 cells were collected. Upon nocodazole wash out cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus OA at 0.5 µM (Noco− OA 0.5), fresh medium plus OA at 1 µM (Noco− OA 1), fresh medium plus nocodazole and OA at 0.5 µM (Noco+ OA 0.5) and fresh medium plus nocodazole and OA at 1 µM (Noco+ OA 1). Nocodazole-treated, prometaphase-arrested ( C ) HeLa and ( D ) hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were divided into two sets, one received vehicle as control (Control) and the other OA at 0.5 µM (OA 0.5). Cells were then taken at the indicated time points of further incubation. Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). The data shown are representative of four independent experiments per type performed under identical conditions and giving similar results.

    Journal: Oncotarget

    Article Title: Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1

    doi: 10.18632/oncotarget.23329

    Figure Lengend Snippet: SAC maintenance and resolution in HeLa and hTERT-RPE1 cells treated with the phosphatase inhibitor Okadaic Acid Nocodazole-treated, prometaphase-arrested, ( A ) HeLa and ( B ) hTERT-RPE1 cells were collected. Upon nocodazole wash out cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus OA at 0.5 µM (Noco− OA 0.5), fresh medium plus OA at 1 µM (Noco− OA 1), fresh medium plus nocodazole and OA at 0.5 µM (Noco+ OA 0.5) and fresh medium plus nocodazole and OA at 1 µM (Noco+ OA 1). Nocodazole-treated, prometaphase-arrested ( C ) HeLa and ( D ) hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were divided into two sets, one received vehicle as control (Control) and the other OA at 0.5 µM (OA 0.5). Cells were then taken at the indicated time points of further incubation. Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). The data shown are representative of four independent experiments per type performed under identical conditions and giving similar results.

    Article Snippet: Prometaphase-arrested cells were obtained by a double thymidine (4 mM; Sigma-Aldrich, St. Louis, MO, USA) block (18 hours each, separated by a 6 hours incubation in fresh medium) followed by release into fresh medium containing nocodazole (500 nM; Calbiochem, Billerica, MA, USA).

    Techniques: Incubation, SDS Page

    PP2A activity does not affect MCC formation and implementation of SAC-dependent mitotic arrest HeLa cells were treated for 18 hours with RO3306 (10 µM). Upon RO3306 wash out, cells were released into: fresh medium (Noco− OA− LB-100-), fresh medium plus nocodazole (Noco+ OA− LB-100-), fresh medium plus nocodazole and LB-100 (Noco+ OA− LB-100+) and fresh medium plus nocodazole and OA (Noco+ OA+ LB-100-). Cell samples were taken at the indicated time points of further incubation and ( A ) Cdc20 Ips (Cdc20 Ip) were resolved on SDS/PAGE and subsequently probed by immunoblotting for the indicated antigens (a total cell lysate, Tot; and mock Ip, Mk Ip, were also included as control in the SDS/PAGE; the asterisk marks immunoglobulin signal, *Ig). ( B ) Total lysates were also probed for the indicated antigens. The data shown are representative of four independent experiments performed under identical conditions and giving similar results.

    Journal: Oncotarget

    Article Title: Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1

    doi: 10.18632/oncotarget.23329

    Figure Lengend Snippet: PP2A activity does not affect MCC formation and implementation of SAC-dependent mitotic arrest HeLa cells were treated for 18 hours with RO3306 (10 µM). Upon RO3306 wash out, cells were released into: fresh medium (Noco− OA− LB-100-), fresh medium plus nocodazole (Noco+ OA− LB-100-), fresh medium plus nocodazole and LB-100 (Noco+ OA− LB-100+) and fresh medium plus nocodazole and OA (Noco+ OA+ LB-100-). Cell samples were taken at the indicated time points of further incubation and ( A ) Cdc20 Ips (Cdc20 Ip) were resolved on SDS/PAGE and subsequently probed by immunoblotting for the indicated antigens (a total cell lysate, Tot; and mock Ip, Mk Ip, were also included as control in the SDS/PAGE; the asterisk marks immunoglobulin signal, *Ig). ( B ) Total lysates were also probed for the indicated antigens. The data shown are representative of four independent experiments performed under identical conditions and giving similar results.

    Article Snippet: Prometaphase-arrested cells were obtained by a double thymidine (4 mM; Sigma-Aldrich, St. Louis, MO, USA) block (18 hours each, separated by a 6 hours incubation in fresh medium) followed by release into fresh medium containing nocodazole (500 nM; Calbiochem, Billerica, MA, USA).

    Techniques: Activity Assay, Incubation, SDS Page

    Effects of the PP2A inhibitor LB-100 on SAC maintenance and resolution in HeLa and hTERT-RPE1 cells Nocodazole-treated, prometaphase-arrested, HeLa cells were collected. Upon nocodazole wash out, cells were divided into two sets, one received vehicle (DMSO) as control and the other LB-100 (10 µM). Cells were then taken at the indicated time points of further incubation. Two cell samples in the control set also received again nocodazole (Noco) and one of them LB-100 (LB-100) and, then, were taken at the indicated time points of further incubation. ( A ) Total samples were separated on SDS/PAGE and immunoblotted for phosphorylated Cdk1 substrates (Cdk1 p-subs), pT481-PRC1, PRC1, cyclin B1 (Cyc B1) and Cdk1. ( B ) Optical pT481-PRC1 (triangles) and cyclin B1 (squares) signal density (arbitrary units; normalized for total PRC1 and Cdk1 optical density values, respectively) were plotted as percent of time 0 samples from control (open symbols) and LB-100-treated (filled symbols) cells. ( C ) Nocodazole-treated, prometaphase-arrested, hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus LB-100 (Noco– LB-100+) or fresh medium plus nocodazole and LB-100 (Noco+ LB-100+). Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). ( D ) Nocodazole-treated, prometaphase-arrested, HeLa cells were collected. Upon nocodazole wash out, cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus 10 µM LB-100 (Noco– LB-100+). Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). The data shown are representative of four independent experiments performed under identical conditions and giving similar results.

    Journal: Oncotarget

    Article Title: Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1

    doi: 10.18632/oncotarget.23329

    Figure Lengend Snippet: Effects of the PP2A inhibitor LB-100 on SAC maintenance and resolution in HeLa and hTERT-RPE1 cells Nocodazole-treated, prometaphase-arrested, HeLa cells were collected. Upon nocodazole wash out, cells were divided into two sets, one received vehicle (DMSO) as control and the other LB-100 (10 µM). Cells were then taken at the indicated time points of further incubation. Two cell samples in the control set also received again nocodazole (Noco) and one of them LB-100 (LB-100) and, then, were taken at the indicated time points of further incubation. ( A ) Total samples were separated on SDS/PAGE and immunoblotted for phosphorylated Cdk1 substrates (Cdk1 p-subs), pT481-PRC1, PRC1, cyclin B1 (Cyc B1) and Cdk1. ( B ) Optical pT481-PRC1 (triangles) and cyclin B1 (squares) signal density (arbitrary units; normalized for total PRC1 and Cdk1 optical density values, respectively) were plotted as percent of time 0 samples from control (open symbols) and LB-100-treated (filled symbols) cells. ( C ) Nocodazole-treated, prometaphase-arrested, hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus LB-100 (Noco– LB-100+) or fresh medium plus nocodazole and LB-100 (Noco+ LB-100+). Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). ( D ) Nocodazole-treated, prometaphase-arrested, HeLa cells were collected. Upon nocodazole wash out, cells were further incubated for 90 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus 10 µM LB-100 (Noco– LB-100+). Total samples were separated on SDS/PAGE and immunoblotted for the indicated antigens. OD, optical density values of above signals (arbitrary units). The data shown are representative of four independent experiments performed under identical conditions and giving similar results.

    Article Snippet: Prometaphase-arrested cells were obtained by a double thymidine (4 mM; Sigma-Aldrich, St. Louis, MO, USA) block (18 hours each, separated by a 6 hours incubation in fresh medium) followed by release into fresh medium containing nocodazole (500 nM; Calbiochem, Billerica, MA, USA).

    Techniques: Incubation, SDS Page

    PP2A activity does not substantially affect progression beyond anaphase Nocodazole-treated, prometaphase-arrested, ( A ) HeLa and ( B ) hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were divided into three sets that received either vehicle as control (CTRL), OA at 0.5 µM (OA), or LB-100. Cells were then taken at the indicated time points of further incubation and spun onto microscopy slides and processed for immunofluorescence staining for α-tubulin and the centromere marker CREST, DNA was stained with Hoechst. Upper graphs: post-anaphase cells were visually scored through microscopy. Error bars refer to variation within three independent experiments performed under identical conditions. Lower photographs: indicative images of HeLa and hTERT-RPE1 cells taken at 60 or 40 min, respectively, of incubation. Scale bars, 10 μm.

    Journal: Oncotarget

    Article Title: Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1

    doi: 10.18632/oncotarget.23329

    Figure Lengend Snippet: PP2A activity does not substantially affect progression beyond anaphase Nocodazole-treated, prometaphase-arrested, ( A ) HeLa and ( B ) hTERT-RPE1 cells were collected. Upon nocodazole wash out, cells were divided into three sets that received either vehicle as control (CTRL), OA at 0.5 µM (OA), or LB-100. Cells were then taken at the indicated time points of further incubation and spun onto microscopy slides and processed for immunofluorescence staining for α-tubulin and the centromere marker CREST, DNA was stained with Hoechst. Upper graphs: post-anaphase cells were visually scored through microscopy. Error bars refer to variation within three independent experiments performed under identical conditions. Lower photographs: indicative images of HeLa and hTERT-RPE1 cells taken at 60 or 40 min, respectively, of incubation. Scale bars, 10 μm.

    Article Snippet: Prometaphase-arrested cells were obtained by a double thymidine (4 mM; Sigma-Aldrich, St. Louis, MO, USA) block (18 hours each, separated by a 6 hours incubation in fresh medium) followed by release into fresh medium containing nocodazole (500 nM; Calbiochem, Billerica, MA, USA).

    Techniques: Activity Assay, Incubation, Microscopy, Immunofluorescence, Staining, Marker

    PP2A activity does not affect MCC maintenance and disassembly Nocodazole-treated, prometaphase-arrested, HeLa cells were washed out of nocodazole and further incubated for 60 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus LB-100 (10 µM; Noco− LB-100+), fresh medium plus OA (0.5 µM; Noco− OA+), fresh medium plus nocodazole and LB-100 (10 µM; Noco+ LB-100+) and fresh medium plus nocodazole and OA (0.5 µM; Noco+ OA+). Cdc20 was immunoprecipitated (Ip) from cell lysates. ( A ) Cdc20 Ips (Cdc20 Ip) were resolved on SDS/PAGE and subsequently probed by immunoblotting for the indicated antigens (a total cell lysate, Tot; and mock Ip, Mk Ip, were also included as control in the SDS/PAGE; the asterisk marks immunoglobulin signal, *Ig). ( B ) Total lysates were also probed for the indicated antigens. ( C ) Nocodazole-treated, prometaphase-arrested, HeLa cells were washed out of nocodazole and further incubated for 60 min in: fresh medium (LB-100- OA−), fresh medium plus LB-100 (10 µM; LB-100+ OA−) or fresh medium plus OA (0.5 µM; LB-100- OA+). BubR1 was probed on blots of long, higher resolving, SDS/PAGE runs of total lysates (short and long blot exposures are shown). Total lysates were also probed for the other indicated antigens. The data shown are representative of three independent experiments performed under identical conditions and giving similar results.

    Journal: Oncotarget

    Article Title: Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1

    doi: 10.18632/oncotarget.23329

    Figure Lengend Snippet: PP2A activity does not affect MCC maintenance and disassembly Nocodazole-treated, prometaphase-arrested, HeLa cells were washed out of nocodazole and further incubated for 60 min in: fresh medium plus nocodazole (Noco+), just fresh medium (Noco−), fresh medium plus LB-100 (10 µM; Noco− LB-100+), fresh medium plus OA (0.5 µM; Noco− OA+), fresh medium plus nocodazole and LB-100 (10 µM; Noco+ LB-100+) and fresh medium plus nocodazole and OA (0.5 µM; Noco+ OA+). Cdc20 was immunoprecipitated (Ip) from cell lysates. ( A ) Cdc20 Ips (Cdc20 Ip) were resolved on SDS/PAGE and subsequently probed by immunoblotting for the indicated antigens (a total cell lysate, Tot; and mock Ip, Mk Ip, were also included as control in the SDS/PAGE; the asterisk marks immunoglobulin signal, *Ig). ( B ) Total lysates were also probed for the indicated antigens. ( C ) Nocodazole-treated, prometaphase-arrested, HeLa cells were washed out of nocodazole and further incubated for 60 min in: fresh medium (LB-100- OA−), fresh medium plus LB-100 (10 µM; LB-100+ OA−) or fresh medium plus OA (0.5 µM; LB-100- OA+). BubR1 was probed on blots of long, higher resolving, SDS/PAGE runs of total lysates (short and long blot exposures are shown). Total lysates were also probed for the other indicated antigens. The data shown are representative of three independent experiments performed under identical conditions and giving similar results.

    Article Snippet: Prometaphase-arrested cells were obtained by a double thymidine (4 mM; Sigma-Aldrich, St. Louis, MO, USA) block (18 hours each, separated by a 6 hours incubation in fresh medium) followed by release into fresh medium containing nocodazole (500 nM; Calbiochem, Billerica, MA, USA).

    Techniques: Activity Assay, Incubation, Immunoprecipitation, SDS Page

    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

    Nocodazole prevents both GLUT4 sorting into the Syntaxin-6-positive perinuclear sub-compartment and insulin-responsive GLUT4 re-exocytosis. (A) Cell surface GLUT4 myc was labeled at 4°C before cells were re-warmed at 37°C for 30 min with or without 3 µM nocodazole. For recovery, nocodazole was washed out after 25 min and replaced with serum free medium for indicated recovery times. Fixed cells were stained for endogenous Stx6 (green). Inset = single optical slice of the perinuclear region. Representative tubulin staining is shown for each condition (far right). (B) Quantification of the co-localization between GLUT4 myc and Stx6 using Pearson's Correlation coefficient (N = 2–4, 10–15 cells per experiment), *p

    Journal: Biology Open

    Article Title: Dynamic GLUT4 sorting through a syntaxin-6 compartment in muscle cells is derailed by insulin resistance-causing ceramide

    doi: 10.1242/bio.20147898

    Figure Lengend Snippet: Nocodazole prevents both GLUT4 sorting into the Syntaxin-6-positive perinuclear sub-compartment and insulin-responsive GLUT4 re-exocytosis. (A) Cell surface GLUT4 myc was labeled at 4°C before cells were re-warmed at 37°C for 30 min with or without 3 µM nocodazole. For recovery, nocodazole was washed out after 25 min and replaced with serum free medium for indicated recovery times. Fixed cells were stained for endogenous Stx6 (green). Inset = single optical slice of the perinuclear region. Representative tubulin staining is shown for each condition (far right). (B) Quantification of the co-localization between GLUT4 myc and Stx6 using Pearson's Correlation coefficient (N = 2–4, 10–15 cells per experiment), *p

    Article Snippet: Nocodazole was purchased from EMD Biosciences Inc. (Darmstadt, Germany) (10 mM stock in DMSO) and C2-ceramide was purchased from Enzo Life Sciences (Farmingdale, NY, USA) (50 mM stock in DMSO).

    Techniques: Labeling, Staining