p y15 cdc2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p y15 cdc2
    P Y15 Cdc2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pe p cdc2 y15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pe p cdc2 y15
    Pe P Cdc2 Y15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti p cdc2 y15 rabbit monoclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti p cdc2 y15 rabbit monoclonal antibody
    Anti P Cdc2 Y15 Rabbit Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti cdc2 p y15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti cdc2 p y15
    (A) Visualization of genomic DNA oxidized in wild-type (WT), TIA1 KO and TIAR KO MEFs by indirect immunofluorescence using an anti-8-oxo-dG antibody. WT MEFs were treated with 100 µM H 2 O 2 for 20 min to promote genomic DNA oxidation as a positive control. Scale bars represent 15 µm. (B) Analysis of cell-cycle phases by flow cytometry after propidium iodide staining. The data are means + SEM (n = 10; * P <0.05; ** P <0.01; *** P <0.001). (C) MEF cells knocked for TIA proteins showed delayed entry into G1/S. The above MEF cells were synchronized at G0/G1 by serum deprivation for 48–96 h. Samples were taken at 0, 16, 18 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (D) MEF cells knocked for TIA proteins showed delayed entry into S. The above MEF cells were synchronized at G1/S by Hydroxyurea blockage for 16–24 h and then released. Samples were taken at 0, 19, 22 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (E) MEF cells knocked for TIA proteins showed delayed entry into G0/G1. The above MEF cells were synchronized at G2/M by Nocodazole blockade for 24–30 h and then released. Samples were taken at 0, 3, 6, 17 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (F) Examples of MEF cells stained with To-Pro-3 (illustrated in green) and phalloidin-TRITC showing impairment of cytokinesis events in TIA1 and TIAR KO MEF. Scale bars represent 20 µm. (G) Analysis of the G2/M DNA damage checkpoint. Immunoblot of total (T), nuclear (N) and cytoplasmic (C) fractions (8 µg) from wild-type (WT), TIA1 KO and TIAR KO MEFs. The blot was probed with antibodies against U2AF65, α-tubulin, <t>Cdc2</t> (total), <t>Cdc2-P</t> <t>(Y15)</t> and cyclin B1 proteins, as indicated. Molecular weight markers and the identities of protein bands are shown.
    Anti Cdc2 P Y15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "T-cell Intracellular Antigen (TIA)-Proteins Deficiency in Murine Embryonic Fibroblasts Alters Cell Cycle Progression and Induces Autophagy"

    Article Title: T-cell Intracellular Antigen (TIA)-Proteins Deficiency in Murine Embryonic Fibroblasts Alters Cell Cycle Progression and Induces Autophagy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075127

    (A) Visualization of genomic DNA oxidized in wild-type (WT), TIA1 KO and TIAR KO MEFs by indirect immunofluorescence using an anti-8-oxo-dG antibody. WT MEFs were treated with 100 µM H 2 O 2 for 20 min to promote genomic DNA oxidation as a positive control. Scale bars represent 15 µm. (B) Analysis of cell-cycle phases by flow cytometry after propidium iodide staining. The data are means + SEM (n = 10; * P <0.05; ** P <0.01; *** P <0.001). (C) MEF cells knocked for TIA proteins showed delayed entry into G1/S. The above MEF cells were synchronized at G0/G1 by serum deprivation for 48–96 h. Samples were taken at 0, 16, 18 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (D) MEF cells knocked for TIA proteins showed delayed entry into S. The above MEF cells were synchronized at G1/S by Hydroxyurea blockage for 16–24 h and then released. Samples were taken at 0, 19, 22 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (E) MEF cells knocked for TIA proteins showed delayed entry into G0/G1. The above MEF cells were synchronized at G2/M by Nocodazole blockade for 24–30 h and then released. Samples were taken at 0, 3, 6, 17 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (F) Examples of MEF cells stained with To-Pro-3 (illustrated in green) and phalloidin-TRITC showing impairment of cytokinesis events in TIA1 and TIAR KO MEF. Scale bars represent 20 µm. (G) Analysis of the G2/M DNA damage checkpoint. Immunoblot of total (T), nuclear (N) and cytoplasmic (C) fractions (8 µg) from wild-type (WT), TIA1 KO and TIAR KO MEFs. The blot was probed with antibodies against U2AF65, α-tubulin, Cdc2 (total), Cdc2-P (Y15) and cyclin B1 proteins, as indicated. Molecular weight markers and the identities of protein bands are shown.
    Figure Legend Snippet: (A) Visualization of genomic DNA oxidized in wild-type (WT), TIA1 KO and TIAR KO MEFs by indirect immunofluorescence using an anti-8-oxo-dG antibody. WT MEFs were treated with 100 µM H 2 O 2 for 20 min to promote genomic DNA oxidation as a positive control. Scale bars represent 15 µm. (B) Analysis of cell-cycle phases by flow cytometry after propidium iodide staining. The data are means + SEM (n = 10; * P <0.05; ** P <0.01; *** P <0.001). (C) MEF cells knocked for TIA proteins showed delayed entry into G1/S. The above MEF cells were synchronized at G0/G1 by serum deprivation for 48–96 h. Samples were taken at 0, 16, 18 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (D) MEF cells knocked for TIA proteins showed delayed entry into S. The above MEF cells were synchronized at G1/S by Hydroxyurea blockage for 16–24 h and then released. Samples were taken at 0, 19, 22 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (E) MEF cells knocked for TIA proteins showed delayed entry into G0/G1. The above MEF cells were synchronized at G2/M by Nocodazole blockade for 24–30 h and then released. Samples were taken at 0, 3, 6, 17 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (F) Examples of MEF cells stained with To-Pro-3 (illustrated in green) and phalloidin-TRITC showing impairment of cytokinesis events in TIA1 and TIAR KO MEF. Scale bars represent 20 µm. (G) Analysis of the G2/M DNA damage checkpoint. Immunoblot of total (T), nuclear (N) and cytoplasmic (C) fractions (8 µg) from wild-type (WT), TIA1 KO and TIAR KO MEFs. The blot was probed with antibodies against U2AF65, α-tubulin, Cdc2 (total), Cdc2-P (Y15) and cyclin B1 proteins, as indicated. Molecular weight markers and the identities of protein bands are shown.

    Techniques Used: Immunofluorescence, Positive Control, Flow Cytometry, Staining, Western Blot, Molecular Weight

    p cdc2 y15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p cdc2 y15
    Targeting AKT3 and WEE1 increased p53 while reducing FOXM1 and <t>CDK1</t> signaling. (A) and (B). Knockdown of WEE1 kinase led to a dose-dependent decrease in the phosphorylation of its substrate CDK1. Dose-dependent increase in the phosphorylation of H2AX was observed. Consequently, this resulted in increased p53, p21 as well as p27 levels, which are known to be inhibitory to cell proliferation. ERK2 served as a control for equal protein loading.
    P Cdc2 Y15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identification of WEE1 as a target to make AKT inhibition more effective in melanoma"

    Article Title: Identification of WEE1 as a target to make AKT inhibition more effective in melanoma

    Journal: Cancer Biology & Therapy

    doi: 10.1080/15384047.2017.1360446

    Targeting AKT3 and WEE1 increased p53 while reducing FOXM1 and CDK1 signaling. (A) and (B). Knockdown of WEE1 kinase led to a dose-dependent decrease in the phosphorylation of its substrate CDK1. Dose-dependent increase in the phosphorylation of H2AX was observed. Consequently, this resulted in increased p53, p21 as well as p27 levels, which are known to be inhibitory to cell proliferation. ERK2 served as a control for equal protein loading.
    Figure Legend Snippet: Targeting AKT3 and WEE1 increased p53 while reducing FOXM1 and CDK1 signaling. (A) and (B). Knockdown of WEE1 kinase led to a dose-dependent decrease in the phosphorylation of its substrate CDK1. Dose-dependent increase in the phosphorylation of H2AX was observed. Consequently, this resulted in increased p53, p21 as well as p27 levels, which are known to be inhibitory to cell proliferation. ERK2 served as a control for equal protein loading.

    Techniques Used:

    Diagram showing the mechanism of synergism for co-targeting AKT and WEE1 signaling pathways. Inhibition of siWEE1 (1) suppresses inhibitory phosphorylation of CDK1 leading to early-G2/M progression. This leads DNA damage (2) and activates p53 signaling. p53 inhibits cell cycle progression by induction of p21, allowing DNA damage repair. If the DNA damage is not repairable, p53 induces apoptosis. However, in many cancer cells, apoptotic cascades are suppressed by oncogenic alterations. Over-activated AKT inhibits pro-apoptotic factors while inducing antiapoptotic factors (3). AKT signaling also enhances cell cycle progression by CyclinD1 mediated phosphorylation of RB (4) and inhibition of p27 (5). Furthermore, AKT phosphorylates and induces Polo-like kinase 1 (PLK1) (6), which in turn inhibits pro-apoptotic functions of p53 and its family members, p63 and p73 (7). In addition, PLK1 also induces FOXM1 activity and M-phase progression (8). Proteins that were validated by Western blotting are shown in bold.
    Figure Legend Snippet: Diagram showing the mechanism of synergism for co-targeting AKT and WEE1 signaling pathways. Inhibition of siWEE1 (1) suppresses inhibitory phosphorylation of CDK1 leading to early-G2/M progression. This leads DNA damage (2) and activates p53 signaling. p53 inhibits cell cycle progression by induction of p21, allowing DNA damage repair. If the DNA damage is not repairable, p53 induces apoptosis. However, in many cancer cells, apoptotic cascades are suppressed by oncogenic alterations. Over-activated AKT inhibits pro-apoptotic factors while inducing antiapoptotic factors (3). AKT signaling also enhances cell cycle progression by CyclinD1 mediated phosphorylation of RB (4) and inhibition of p27 (5). Furthermore, AKT phosphorylates and induces Polo-like kinase 1 (PLK1) (6), which in turn inhibits pro-apoptotic functions of p53 and its family members, p63 and p73 (7). In addition, PLK1 also induces FOXM1 activity and M-phase progression (8). Proteins that were validated by Western blotting are shown in bold.

    Techniques Used: Inhibition, Activity Assay, Western Blot

    p cdc2 y15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p cdc2 y15
    Targeting AKT3 and WEE1 increased p53 while reducing FOXM1 and <t>CDK1</t> signaling. (A) and (B). Knockdown of WEE1 kinase led to a dose-dependent decrease in the phosphorylation of its substrate CDK1. Dose-dependent increase in the phosphorylation of H2AX was observed. Consequently, this resulted in increased p53, p21 as well as p27 levels, which are known to be inhibitory to cell proliferation. ERK2 served as a control for equal protein loading.
    P Cdc2 Y15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p cdc2 y15/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p cdc2 y15 - by Bioz Stars, 2024-07
    96/100 stars

    Images

    1) Product Images from "Identification of WEE1 as a target to make AKT inhibition more effective in melanoma"

    Article Title: Identification of WEE1 as a target to make AKT inhibition more effective in melanoma

    Journal: Cancer Biology & Therapy

    doi: 10.1080/15384047.2017.1360446

    Targeting AKT3 and WEE1 increased p53 while reducing FOXM1 and CDK1 signaling. (A) and (B). Knockdown of WEE1 kinase led to a dose-dependent decrease in the phosphorylation of its substrate CDK1. Dose-dependent increase in the phosphorylation of H2AX was observed. Consequently, this resulted in increased p53, p21 as well as p27 levels, which are known to be inhibitory to cell proliferation. ERK2 served as a control for equal protein loading.
    Figure Legend Snippet: Targeting AKT3 and WEE1 increased p53 while reducing FOXM1 and CDK1 signaling. (A) and (B). Knockdown of WEE1 kinase led to a dose-dependent decrease in the phosphorylation of its substrate CDK1. Dose-dependent increase in the phosphorylation of H2AX was observed. Consequently, this resulted in increased p53, p21 as well as p27 levels, which are known to be inhibitory to cell proliferation. ERK2 served as a control for equal protein loading.

    Techniques Used:

    Diagram showing the mechanism of synergism for co-targeting AKT and WEE1 signaling pathways. Inhibition of siWEE1 (1) suppresses inhibitory phosphorylation of CDK1 leading to early-G2/M progression. This leads DNA damage (2) and activates p53 signaling. p53 inhibits cell cycle progression by induction of p21, allowing DNA damage repair. If the DNA damage is not repairable, p53 induces apoptosis. However, in many cancer cells, apoptotic cascades are suppressed by oncogenic alterations. Over-activated AKT inhibits pro-apoptotic factors while inducing antiapoptotic factors (3). AKT signaling also enhances cell cycle progression by CyclinD1 mediated phosphorylation of RB (4) and inhibition of p27 (5). Furthermore, AKT phosphorylates and induces Polo-like kinase 1 (PLK1) (6), which in turn inhibits pro-apoptotic functions of p53 and its family members, p63 and p73 (7). In addition, PLK1 also induces FOXM1 activity and M-phase progression (8). Proteins that were validated by Western blotting are shown in bold.
    Figure Legend Snippet: Diagram showing the mechanism of synergism for co-targeting AKT and WEE1 signaling pathways. Inhibition of siWEE1 (1) suppresses inhibitory phosphorylation of CDK1 leading to early-G2/M progression. This leads DNA damage (2) and activates p53 signaling. p53 inhibits cell cycle progression by induction of p21, allowing DNA damage repair. If the DNA damage is not repairable, p53 induces apoptosis. However, in many cancer cells, apoptotic cascades are suppressed by oncogenic alterations. Over-activated AKT inhibits pro-apoptotic factors while inducing antiapoptotic factors (3). AKT signaling also enhances cell cycle progression by CyclinD1 mediated phosphorylation of RB (4) and inhibition of p27 (5). Furthermore, AKT phosphorylates and induces Polo-like kinase 1 (PLK1) (6), which in turn inhibits pro-apoptotic functions of p53 and its family members, p63 and p73 (7). In addition, PLK1 also induces FOXM1 activity and M-phase progression (8). Proteins that were validated by Western blotting are shown in bold.

    Techniques Used: Inhibition, Activity Assay, Western Blot

    cdc2 y15 p  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cdc2 y15 p
    Depletion of slp1 arrests cells in metaphase but cannot prevent cytokinesis. (A) Overview of the experimental setup used to repress the expression of slp1 under the control of p41-nmt1 . Slp1 is repressed in the presence of 15 µM thiamine (+T) and expressed in the absence of thiamine (−T). Wild type cells were used as control for the endogenous expression of slp1. (B,D–F) cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for mRNA expression, microscopy, and western blot. (B) mRNA expression of slp1 in wild type and p41-nmt1:slp1 cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of four biological replicates are shown. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test. ns: p > 0.05; ****: p ≤ 0.0001. (C) Image of wild type andp41- nmt1:slp1 cells streaked on EMM agar plates that contain 15 µM thiamine or not at 30°C. (D) Images of ethanol-fixed cells stained with DAPI to visualise DNA compactation and blankophor to visualise cell wall and septum by microscopy. In the lower panels, Differently-colored arrowheads indicate the different phenotypes scored in (E) : Cycling cells (red), metaphase-arrested cells with condensed chromosomes (cyan), cut cells (purple) and septated cells with unequal segregation of chromosomes (yellow). Scale bar 5 µm. (E) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (F) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and <t>Cdc2</t> phosphorylated at <t>Y15</t> served as indicators of CDK activity and were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2.
    Cdc2 Y15 P, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdc2 y15 p/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cdc2 y15 p - by Bioz Stars, 2024-07
    96/100 stars

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    1) Product Images from "Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast"

    Article Title: Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2022.876810

    Depletion of slp1 arrests cells in metaphase but cannot prevent cytokinesis. (A) Overview of the experimental setup used to repress the expression of slp1 under the control of p41-nmt1 . Slp1 is repressed in the presence of 15 µM thiamine (+T) and expressed in the absence of thiamine (−T). Wild type cells were used as control for the endogenous expression of slp1. (B,D–F) cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for mRNA expression, microscopy, and western blot. (B) mRNA expression of slp1 in wild type and p41-nmt1:slp1 cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of four biological replicates are shown. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test. ns: p > 0.05; ****: p ≤ 0.0001. (C) Image of wild type andp41- nmt1:slp1 cells streaked on EMM agar plates that contain 15 µM thiamine or not at 30°C. (D) Images of ethanol-fixed cells stained with DAPI to visualise DNA compactation and blankophor to visualise cell wall and septum by microscopy. In the lower panels, Differently-colored arrowheads indicate the different phenotypes scored in (E) : Cycling cells (red), metaphase-arrested cells with condensed chromosomes (cyan), cut cells (purple) and septated cells with unequal segregation of chromosomes (yellow). Scale bar 5 µm. (E) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (F) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 served as indicators of CDK activity and were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2.
    Figure Legend Snippet: Depletion of slp1 arrests cells in metaphase but cannot prevent cytokinesis. (A) Overview of the experimental setup used to repress the expression of slp1 under the control of p41-nmt1 . Slp1 is repressed in the presence of 15 µM thiamine (+T) and expressed in the absence of thiamine (−T). Wild type cells were used as control for the endogenous expression of slp1. (B,D–F) cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for mRNA expression, microscopy, and western blot. (B) mRNA expression of slp1 in wild type and p41-nmt1:slp1 cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of four biological replicates are shown. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test. ns: p > 0.05; ****: p ≤ 0.0001. (C) Image of wild type andp41- nmt1:slp1 cells streaked on EMM agar plates that contain 15 µM thiamine or not at 30°C. (D) Images of ethanol-fixed cells stained with DAPI to visualise DNA compactation and blankophor to visualise cell wall and septum by microscopy. In the lower panels, Differently-colored arrowheads indicate the different phenotypes scored in (E) : Cycling cells (red), metaphase-arrested cells with condensed chromosomes (cyan), cut cells (purple) and septated cells with unequal segregation of chromosomes (yellow). Scale bar 5 µm. (E) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (F) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 served as indicators of CDK activity and were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2.

    Techniques Used: Expressing, Microscopy, Western Blot, Staining, Activity Assay

    Protein phosphatases influence the mitotic arrest. (A–F) cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A,B) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Y15-Cdc2 phosphorylation in p41-nmt1:slp1 and p41-nmt1:slp1 clp1Δ (A) or p41-nmt1:slp1 and p41-nmt1:slp1 ppa2Δ strains (B) were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (C,D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E,F) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C,D) . Scale bar 5 µm.
    Figure Legend Snippet: Protein phosphatases influence the mitotic arrest. (A–F) cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A,B) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Y15-Cdc2 phosphorylation in p41-nmt1:slp1 and p41-nmt1:slp1 clp1Δ (A) or p41-nmt1:slp1 and p41-nmt1:slp1 ppa2Δ strains (B) were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (C,D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E,F) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C,D) . Scale bar 5 µm.

    Techniques Used: Microscopy, Western Blot, Staining

    B55 Pab1 and B56 Par1 PP2A-regulatory subunits have opposite effects in the induction of septation in metaphase arrested cells. (A–C) The indicated strains were grown in EMM and then shifted to EMM with 0.5 mM 1-Naphthaleneacetic acid (auxin) for Pab1 degradation and/or 15 µm thiamine ( 41nmt promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A) Depletion of Pab1 was detected by western blot against its N-terminal 3PK tag. See also . Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 served as indicators of CDK activity and were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (B) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (C) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells of p41-nmt1:slp1p41-nmt1-3PK-miniAID-pab1 in (B) . Scale bar 5 µm. (D–F) p41 - nmt1:slp1 par1Δ cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells of p41-nmt1:slp1 par1Δ in (D) . Scale bar 5 µm. (F) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 in p41-nmt1:slp1 and p41-nmt1:slp1 par1Δ strains were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2.
    Figure Legend Snippet: B55 Pab1 and B56 Par1 PP2A-regulatory subunits have opposite effects in the induction of septation in metaphase arrested cells. (A–C) The indicated strains were grown in EMM and then shifted to EMM with 0.5 mM 1-Naphthaleneacetic acid (auxin) for Pab1 degradation and/or 15 µm thiamine ( 41nmt promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A) Depletion of Pab1 was detected by western blot against its N-terminal 3PK tag. See also . Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 served as indicators of CDK activity and were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (B) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (C) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells of p41-nmt1:slp1p41-nmt1-3PK-miniAID-pab1 in (B) . Scale bar 5 µm. (D–F) p41 - nmt1:slp1 par1Δ cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells of p41-nmt1:slp1 par1Δ in (D) . Scale bar 5 µm. (F) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 in p41-nmt1:slp1 and p41-nmt1:slp1 par1Δ strains were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2.

    Techniques Used: Microscopy, Western Blot, Activity Assay, Staining

    Interplay between CDK and PP2A-B55 Pab1 in the regulation of septation. (A–F) Cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A,B) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 in p41-nmt1:slp1 , p41-nmt1:slp1 par1Δ and p41-nmt1:slp1 cdc2-3w par1Δ (A) or p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ strains (B) were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (C,D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) in p41-nmt1:slp1 cdc2-3w par1Δ (C) and p41-nmt1:slp1 igo1Δ (D) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E,F) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C,D) . Scale bar 5 µm.
    Figure Legend Snippet: Interplay between CDK and PP2A-B55 Pab1 in the regulation of septation. (A–F) Cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A,B) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 in p41-nmt1:slp1 , p41-nmt1:slp1 par1Δ and p41-nmt1:slp1 cdc2-3w par1Δ (A) or p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ strains (B) were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (C,D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) in p41-nmt1:slp1 cdc2-3w par1Δ (C) and p41-nmt1:slp1 igo1Δ (D) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E,F) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C,D) . Scale bar 5 µm.

    Techniques Used: Microscopy, Western Blot, Staining

    Dynamic Byr4 phosphorylation and Cdc13 localisation during a sustained metaphase arrest. (A–D) Cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. For the nda3-3KM311 mutant, cells were grown at 30 and then shifted to 18°C for 6 h. (A,C) Byr4 levels and phosphorylation (band shift) were detected by western blot in p41-nmt1:slp1 , p41-nmt1:slp1 ppa2Δ , p41-nmt1:slp1 par1Δ and nda3-3KM311 mutant (A) ; p41-nmt1:slp1 , p41-nmt1:slp1 par1Δ and p41-nmt1:slp1 cdc2-3w par1Δ (B) and p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ (B) or p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ strains (C) in EMM -T and EMM +T at the indicated time points. Cdc2 (PSTAIR) was used as a protein loading control. (D) Representative images of cdc13mCherry localization and Blankophor-stained ethanol-fixed cells upon slp1 repression by thiamine. Scale bar 5 µm.
    Figure Legend Snippet: Dynamic Byr4 phosphorylation and Cdc13 localisation during a sustained metaphase arrest. (A–D) Cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. For the nda3-3KM311 mutant, cells were grown at 30 and then shifted to 18°C for 6 h. (A,C) Byr4 levels and phosphorylation (band shift) were detected by western blot in p41-nmt1:slp1 , p41-nmt1:slp1 ppa2Δ , p41-nmt1:slp1 par1Δ and nda3-3KM311 mutant (A) ; p41-nmt1:slp1 , p41-nmt1:slp1 par1Δ and p41-nmt1:slp1 cdc2-3w par1Δ (B) and p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ (B) or p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ strains (C) in EMM -T and EMM +T at the indicated time points. Cdc2 (PSTAIR) was used as a protein loading control. (D) Representative images of cdc13mCherry localization and Blankophor-stained ethanol-fixed cells upon slp1 repression by thiamine. Scale bar 5 µm.

    Techniques Used: Microscopy, Western Blot, Mutagenesis, Electrophoretic Mobility Shift Assay, Staining

    The mitotic transcriptional wave influences the behaviour of cells arrested in metaphase. (A–D) Cells were grown exponentially in EMM at 30°C or 25°C ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C or 36°C. Samples were collected at the indicated time points for mRNA expression and microscopy. (A) mRNA expression of slp1, ace2, gas1 and par2 in wild type and in p41-nmt1:slp1 or p41-nmt1:slp1 par1Δ cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of three biological replicate are shown. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test. ns: p > 0.05; *: p ≤ 0.05; ** ≤ 0.01; ****: p ≤ 0.0001. (B) Left panel. mRNA expression of ace2 in p41-nmt1:slp1 or p41-nmt1:slp1 par1Δ , p41-nmt1:slp1 cdc2-3w par1Δ and p41-nmt1:slp1 igo1Δ cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of at least two biological replicates are shown. Right panel. mRNA expression of ace2 in p41-nmt1:slp1 or p41-nmt1:slp1 sak1ts cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of at least two biological replicates are shown. (C) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) in p41-nmt1:slp1 (left panel) and p41-nmt1:slp1 sak1ts (right panel) at each indicated time point after thiamine addition. (*) cells grown at 25°C. At least 200 cells were counted per time point. (D) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C) . Scale bar 5 µm.
    Figure Legend Snippet: The mitotic transcriptional wave influences the behaviour of cells arrested in metaphase. (A–D) Cells were grown exponentially in EMM at 30°C or 25°C ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C or 36°C. Samples were collected at the indicated time points for mRNA expression and microscopy. (A) mRNA expression of slp1, ace2, gas1 and par2 in wild type and in p41-nmt1:slp1 or p41-nmt1:slp1 par1Δ cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of three biological replicate are shown. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test. ns: p > 0.05; *: p ≤ 0.05; ** ≤ 0.01; ****: p ≤ 0.0001. (B) Left panel. mRNA expression of ace2 in p41-nmt1:slp1 or p41-nmt1:slp1 par1Δ , p41-nmt1:slp1 cdc2-3w par1Δ and p41-nmt1:slp1 igo1Δ cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of at least two biological replicates are shown. Right panel. mRNA expression of ace2 in p41-nmt1:slp1 or p41-nmt1:slp1 sak1ts cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of at least two biological replicates are shown. (C) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) in p41-nmt1:slp1 (left panel) and p41-nmt1:slp1 sak1ts (right panel) at each indicated time point after thiamine addition. (*) cells grown at 25°C. At least 200 cells were counted per time point. (D) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C) . Scale bar 5 µm.

    Techniques Used: Expressing, Microscopy, Staining

    Proposed model for the regulation of cytokinesis by PP2A phosphatases and mitotic CDK complex during a prolonged mitotic arrest. (A) Mitotic arrest was induced through transcriptional repression of the APC/C activator Slp1 (under the control of the thiamine-repressible promoter p41-nmt1 ). Silencing of slp1 resulted in the rapid accumulation of the mitotic cyclin Cdc13, increased mitotic phosphorylation and chromosome condensation. Upon prolonged periods of mitotic arrest, mitotic dephosphorylation and induction of cytokinesis could be observed in the presence of condensed chromosomes and high levels of Cdc13. (B) Mitotic CDK both favors and represses cytokinesis by inactivating the Spg1 GAP (activation of the top of the SIN cascade), while preventing recruitment of Sid1:Cdc14 (repression of the bottom of the SIN cascade) ( ; ; ). PP2A-B55 Pab1 and PP2A-B56 Par1 have opposite effects in the regulation of cytokinesis: PP2A-B55 Pab1 represses cytokinesis and, in its absence, untimely septation in the presence of unsegregated chromosomes occurs at high frequency in mitotically-arrested cells (1). The mitotic CDK complex establishes a double negative feedback loop with PP2A-B55 Pab1 through the Ppk18-Igo1 (Greatwall-ENSA) pathway (2). In consequence, high CDK activity during a prolonged mitotic arrest would result in PP2A-B55 Pab1 inactivation and induction of cytokinesis. In our experimental conditions, engagement of this double negative feedback loop would be sufficient to overcome the repression of cytokinesis by CDK. PP2A-B56 Par1 favors cytokinesis through different means: it participates in the activation of the mitotic CDK complex at the G2/M transition (3). In its absence, cells enter mitosis with residual Cdc2-Tyr15 phosphorylation. Presumably, this hinders the repression of PP2A-B55 Pab1 by CDK and results in septation being prevented. The underlying mechanism of this regulation is unknown at present, but might involve modulation of Wee1 and/or Cdc25 activities. In addition, PP2A-B56 Par1 participates in the activation of the mitotic transcriptional program brought about by the RFX transcription factor Sak1 (4). Loss of either Sak1 or PP2A-B56 Par1 impairs the expression of cytokinetic regulators and this contributes to the absence of cytokinesis during the arrest in these mutants. (C) Steady mitotic dephosphorylation during the arrest requires the activities of Clp1, PP2A-B55 Pab1 and PP2A-B56 Par1 .
    Figure Legend Snippet: Proposed model for the regulation of cytokinesis by PP2A phosphatases and mitotic CDK complex during a prolonged mitotic arrest. (A) Mitotic arrest was induced through transcriptional repression of the APC/C activator Slp1 (under the control of the thiamine-repressible promoter p41-nmt1 ). Silencing of slp1 resulted in the rapid accumulation of the mitotic cyclin Cdc13, increased mitotic phosphorylation and chromosome condensation. Upon prolonged periods of mitotic arrest, mitotic dephosphorylation and induction of cytokinesis could be observed in the presence of condensed chromosomes and high levels of Cdc13. (B) Mitotic CDK both favors and represses cytokinesis by inactivating the Spg1 GAP (activation of the top of the SIN cascade), while preventing recruitment of Sid1:Cdc14 (repression of the bottom of the SIN cascade) ( ; ; ). PP2A-B55 Pab1 and PP2A-B56 Par1 have opposite effects in the regulation of cytokinesis: PP2A-B55 Pab1 represses cytokinesis and, in its absence, untimely septation in the presence of unsegregated chromosomes occurs at high frequency in mitotically-arrested cells (1). The mitotic CDK complex establishes a double negative feedback loop with PP2A-B55 Pab1 through the Ppk18-Igo1 (Greatwall-ENSA) pathway (2). In consequence, high CDK activity during a prolonged mitotic arrest would result in PP2A-B55 Pab1 inactivation and induction of cytokinesis. In our experimental conditions, engagement of this double negative feedback loop would be sufficient to overcome the repression of cytokinesis by CDK. PP2A-B56 Par1 favors cytokinesis through different means: it participates in the activation of the mitotic CDK complex at the G2/M transition (3). In its absence, cells enter mitosis with residual Cdc2-Tyr15 phosphorylation. Presumably, this hinders the repression of PP2A-B55 Pab1 by CDK and results in septation being prevented. The underlying mechanism of this regulation is unknown at present, but might involve modulation of Wee1 and/or Cdc25 activities. In addition, PP2A-B56 Par1 participates in the activation of the mitotic transcriptional program brought about by the RFX transcription factor Sak1 (4). Loss of either Sak1 or PP2A-B56 Par1 impairs the expression of cytokinetic regulators and this contributes to the absence of cytokinesis during the arrest in these mutants. (C) Steady mitotic dephosphorylation during the arrest requires the activities of Clp1, PP2A-B55 Pab1 and PP2A-B56 Par1 .

    Techniques Used: De-Phosphorylation Assay, Activation Assay, Activity Assay, Expressing

    anti p y15 cdk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti p y15 cdk1
    Anti P Y15 Cdk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti p cdc2 y15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti p cdc2 y15
    ( A ) Cell cycle profiles of control and BI-D1870-treated HL60 cells by flow cytometry. HL60 cells were treated with BI-D1870 (5 μM). Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and antibodies. Bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (p-H3) was detected by multi-parameter flow cytometry. Percentages of cell populations at each cell cycle phase determined by DNA content (DAPI) and levels of p-H3, Cyclin A and Cyclin B were graphed. ( B ) Protein levels of G2/M phase markers (p-Rb (S780), <t>p-CDC2</t> <t>(Y15),</t> Cyclin B and Cyclin A), apoptosis marker (Cleaved Caspase 3) and phosphorylated Ribosomal protein S6 (p-RPS6 (S235/236)) in BI-D1870-treated HL60 cells. Cell extracts were prepared at the indicated times after BI-D1870 treatment. β-Actin was used as an internal control. ( C ) Accumulation of metaphase cells following the treatment of BI-D1870. After treatment with BI-D1870 (5 μM) for the indicated times, HL60 cells were fixed and stained with DAPI and antibodies. Mitotic phases were further characterized in p-H3-positive populations by measuring the levels of Cyclin A and Cyclin B. Data represent the percentages of cell populations residing at each mitotic phase analyzed by the levels of Cyclin A and Cyclin B in the mitotic population. ( D ) Induction of mitotic arrest in KG1 cells by BI-D1870 treatment. KG1 cells were treated with BI-D1870 (5 μM) for indicated times. Cells were fixed and stained with DAPI and antibodies against p-H3, Cyclin A, and Cyclin B. Cell cycle profiles of DMSO or compound-treated KG1 cells were shown as the bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (top). Each mitotic phase distribution was identified as the cellular expression of Cyclin A and Cyclin B in mitotic cells (bottom). The percentage cell population at each cell cycle stage is shown. Flow cytometric profiles represent one out of three experiments with similar results. Data are graphed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.
    Anti P Cdc2 Y15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p cdc2 y15/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
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    1) Product Images from "RSK inhibitor BI-D1870 inhibits acute myeloid leukemia cell proliferation by targeting mitotic exit"

    Article Title: RSK inhibitor BI-D1870 inhibits acute myeloid leukemia cell proliferation by targeting mitotic exit

    Journal: Oncotarget

    doi: 10.18632/oncotarget.27630

    ( A ) Cell cycle profiles of control and BI-D1870-treated HL60 cells by flow cytometry. HL60 cells were treated with BI-D1870 (5 μM). Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and antibodies. Bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (p-H3) was detected by multi-parameter flow cytometry. Percentages of cell populations at each cell cycle phase determined by DNA content (DAPI) and levels of p-H3, Cyclin A and Cyclin B were graphed. ( B ) Protein levels of G2/M phase markers (p-Rb (S780), p-CDC2 (Y15), Cyclin B and Cyclin A), apoptosis marker (Cleaved Caspase 3) and phosphorylated Ribosomal protein S6 (p-RPS6 (S235/236)) in BI-D1870-treated HL60 cells. Cell extracts were prepared at the indicated times after BI-D1870 treatment. β-Actin was used as an internal control. ( C ) Accumulation of metaphase cells following the treatment of BI-D1870. After treatment with BI-D1870 (5 μM) for the indicated times, HL60 cells were fixed and stained with DAPI and antibodies. Mitotic phases were further characterized in p-H3-positive populations by measuring the levels of Cyclin A and Cyclin B. Data represent the percentages of cell populations residing at each mitotic phase analyzed by the levels of Cyclin A and Cyclin B in the mitotic population. ( D ) Induction of mitotic arrest in KG1 cells by BI-D1870 treatment. KG1 cells were treated with BI-D1870 (5 μM) for indicated times. Cells were fixed and stained with DAPI and antibodies against p-H3, Cyclin A, and Cyclin B. Cell cycle profiles of DMSO or compound-treated KG1 cells were shown as the bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (top). Each mitotic phase distribution was identified as the cellular expression of Cyclin A and Cyclin B in mitotic cells (bottom). The percentage cell population at each cell cycle stage is shown. Flow cytometric profiles represent one out of three experiments with similar results. Data are graphed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.
    Figure Legend Snippet: ( A ) Cell cycle profiles of control and BI-D1870-treated HL60 cells by flow cytometry. HL60 cells were treated with BI-D1870 (5 μM). Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and antibodies. Bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (p-H3) was detected by multi-parameter flow cytometry. Percentages of cell populations at each cell cycle phase determined by DNA content (DAPI) and levels of p-H3, Cyclin A and Cyclin B were graphed. ( B ) Protein levels of G2/M phase markers (p-Rb (S780), p-CDC2 (Y15), Cyclin B and Cyclin A), apoptosis marker (Cleaved Caspase 3) and phosphorylated Ribosomal protein S6 (p-RPS6 (S235/236)) in BI-D1870-treated HL60 cells. Cell extracts were prepared at the indicated times after BI-D1870 treatment. β-Actin was used as an internal control. ( C ) Accumulation of metaphase cells following the treatment of BI-D1870. After treatment with BI-D1870 (5 μM) for the indicated times, HL60 cells were fixed and stained with DAPI and antibodies. Mitotic phases were further characterized in p-H3-positive populations by measuring the levels of Cyclin A and Cyclin B. Data represent the percentages of cell populations residing at each mitotic phase analyzed by the levels of Cyclin A and Cyclin B in the mitotic population. ( D ) Induction of mitotic arrest in KG1 cells by BI-D1870 treatment. KG1 cells were treated with BI-D1870 (5 μM) for indicated times. Cells were fixed and stained with DAPI and antibodies against p-H3, Cyclin A, and Cyclin B. Cell cycle profiles of DMSO or compound-treated KG1 cells were shown as the bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (top). Each mitotic phase distribution was identified as the cellular expression of Cyclin A and Cyclin B in mitotic cells (bottom). The percentage cell population at each cell cycle stage is shown. Flow cytometric profiles represent one out of three experiments with similar results. Data are graphed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.

    Techniques Used: Flow Cytometry, Staining, Marker, Expressing

    HL60 cells were treated with BI-D1870 (5 μM) for the indicated hours, and then cells were collected and analyzed for the cellular levels of p-CDC25C (S198) ( A ) or p-CDC2 (Y15) ( B ). Fixed cells were stained with DAPI and antibodies against Cyclin A, Cyclin B, p-H3, p-CDC2 (Y15), and p-CDC25C (S198). (A) Decrease in positive phosphorylation at serine 198 (S198) on CDC25C following the treatment of BI-D1870. Representative plot of p-CDC25C (S198) levels in G2 and mitotic phases populations following the treatment of BI-D1870. The graph shows the MFI of p-CDC25C (S198) in G2 and mitotic phases populations of HL60 cells treated with or without BI-D1870. (B) Temporary inhibition of CDC2 activation in the G2 phase by BI-D1870 treatment. CDC2 activity is regulated in a negative fashion by phosphorylation at tyrosine 15 (Y15). Representative flow cytometric profile of p-CDC2 (Y15) levels in the G2 and mitotic phases populations following the treatment of BI-D1870. The graph shows median fluorescence intensities (MFI) of p-CDC2 (Y15) in the G2 and mitotic phases populations of HL60 cells treated with or without BI-D1870. Negative phosphorylation at Y15 on CDC2 was transiently enhanced in only the G2 phase at 2 h after BI-D1870 treatment. Flow cytometric profiles represent one out of three independent experiments. Values are graphed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01.
    Figure Legend Snippet: HL60 cells were treated with BI-D1870 (5 μM) for the indicated hours, and then cells were collected and analyzed for the cellular levels of p-CDC25C (S198) ( A ) or p-CDC2 (Y15) ( B ). Fixed cells were stained with DAPI and antibodies against Cyclin A, Cyclin B, p-H3, p-CDC2 (Y15), and p-CDC25C (S198). (A) Decrease in positive phosphorylation at serine 198 (S198) on CDC25C following the treatment of BI-D1870. Representative plot of p-CDC25C (S198) levels in G2 and mitotic phases populations following the treatment of BI-D1870. The graph shows the MFI of p-CDC25C (S198) in G2 and mitotic phases populations of HL60 cells treated with or without BI-D1870. (B) Temporary inhibition of CDC2 activation in the G2 phase by BI-D1870 treatment. CDC2 activity is regulated in a negative fashion by phosphorylation at tyrosine 15 (Y15). Representative flow cytometric profile of p-CDC2 (Y15) levels in the G2 and mitotic phases populations following the treatment of BI-D1870. The graph shows median fluorescence intensities (MFI) of p-CDC2 (Y15) in the G2 and mitotic phases populations of HL60 cells treated with or without BI-D1870. Negative phosphorylation at Y15 on CDC2 was transiently enhanced in only the G2 phase at 2 h after BI-D1870 treatment. Flow cytometric profiles represent one out of three independent experiments. Values are graphed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01.

    Techniques Used: Staining, Inhibition, Activation Assay, Activity Assay, Fluorescence

    p cdc2 p cdk1 y15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p cdc2 p cdk1 y15
    P Cdc2 P Cdk1 Y15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Visualization of genomic DNA oxidized in wild-type (WT), TIA1 KO and TIAR KO MEFs by indirect immunofluorescence using an anti-8-oxo-dG antibody. WT MEFs were treated with 100 µM H 2 O 2 for 20 min to promote genomic DNA oxidation as a positive control. Scale bars represent 15 µm. (B) Analysis of cell-cycle phases by flow cytometry after propidium iodide staining. The data are means + SEM (n = 10; * P <0.05; ** P <0.01; *** P <0.001). (C) MEF cells knocked for TIA proteins showed delayed entry into G1/S. The above MEF cells were synchronized at G0/G1 by serum deprivation for 48–96 h. Samples were taken at 0, 16, 18 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (D) MEF cells knocked for TIA proteins showed delayed entry into S. The above MEF cells were synchronized at G1/S by Hydroxyurea blockage for 16–24 h and then released. Samples were taken at 0, 19, 22 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (E) MEF cells knocked for TIA proteins showed delayed entry into G0/G1. The above MEF cells were synchronized at G2/M by Nocodazole blockade for 24–30 h and then released. Samples were taken at 0, 3, 6, 17 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (F) Examples of MEF cells stained with To-Pro-3 (illustrated in green) and phalloidin-TRITC showing impairment of cytokinesis events in TIA1 and TIAR KO MEF. Scale bars represent 20 µm. (G) Analysis of the G2/M DNA damage checkpoint. Immunoblot of total (T), nuclear (N) and cytoplasmic (C) fractions (8 µg) from wild-type (WT), TIA1 KO and TIAR KO MEFs. The blot was probed with antibodies against U2AF65, α-tubulin, <t>Cdc2</t> (total), <t>Cdc2-P</t> <t>(Y15)</t> and cyclin B1 proteins, as indicated. Molecular weight markers and the identities of protein bands are shown.
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    Targeting AKT3 and WEE1 increased p53 while reducing FOXM1 and <t>CDK1</t> signaling. (A) and (B). Knockdown of WEE1 kinase led to a dose-dependent decrease in the phosphorylation of its substrate CDK1. Dose-dependent increase in the phosphorylation of H2AX was observed. Consequently, this resulted in increased p53, p21 as well as p27 levels, which are known to be inhibitory to cell proliferation. ERK2 served as a control for equal protein loading.
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    Depletion of slp1 arrests cells in metaphase but cannot prevent cytokinesis. (A) Overview of the experimental setup used to repress the expression of slp1 under the control of p41-nmt1 . Slp1 is repressed in the presence of 15 µM thiamine (+T) and expressed in the absence of thiamine (−T). Wild type cells were used as control for the endogenous expression of slp1. (B,D–F) cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for mRNA expression, microscopy, and western blot. (B) mRNA expression of slp1 in wild type and p41-nmt1:slp1 cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of four biological replicates are shown. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test. ns: p > 0.05; ****: p ≤ 0.0001. (C) Image of wild type andp41- nmt1:slp1 cells streaked on EMM agar plates that contain 15 µM thiamine or not at 30°C. (D) Images of ethanol-fixed cells stained with DAPI to visualise DNA compactation and blankophor to visualise cell wall and septum by microscopy. In the lower panels, Differently-colored arrowheads indicate the different phenotypes scored in (E) : Cycling cells (red), metaphase-arrested cells with condensed chromosomes (cyan), cut cells (purple) and septated cells with unequal segregation of chromosomes (yellow). Scale bar 5 µm. (E) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (F) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and <t>Cdc2</t> phosphorylated at <t>Y15</t> served as indicators of CDK activity and were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2.
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    Depletion of slp1 arrests cells in metaphase but cannot prevent cytokinesis. (A) Overview of the experimental setup used to repress the expression of slp1 under the control of p41-nmt1 . Slp1 is repressed in the presence of 15 µM thiamine (+T) and expressed in the absence of thiamine (−T). Wild type cells were used as control for the endogenous expression of slp1. (B,D–F) cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for mRNA expression, microscopy, and western blot. (B) mRNA expression of slp1 in wild type and p41-nmt1:slp1 cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of four biological replicates are shown. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test. ns: p > 0.05; ****: p ≤ 0.0001. (C) Image of wild type andp41- nmt1:slp1 cells streaked on EMM agar plates that contain 15 µM thiamine or not at 30°C. (D) Images of ethanol-fixed cells stained with DAPI to visualise DNA compactation and blankophor to visualise cell wall and septum by microscopy. In the lower panels, Differently-colored arrowheads indicate the different phenotypes scored in (E) : Cycling cells (red), metaphase-arrested cells with condensed chromosomes (cyan), cut cells (purple) and septated cells with unequal segregation of chromosomes (yellow). Scale bar 5 µm. (E) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (F) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and <t>Cdc2</t> phosphorylated at <t>Y15</t> served as indicators of CDK activity and were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2.
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    ( A ) Cell cycle profiles of control and BI-D1870-treated HL60 cells by flow cytometry. HL60 cells were treated with BI-D1870 (5 μM). Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and antibodies. Bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (p-H3) was detected by multi-parameter flow cytometry. Percentages of cell populations at each cell cycle phase determined by DNA content (DAPI) and levels of p-H3, Cyclin A and Cyclin B were graphed. ( B ) Protein levels of G2/M phase markers (p-Rb (S780), <t>p-CDC2</t> <t>(Y15),</t> Cyclin B and Cyclin A), apoptosis marker (Cleaved Caspase 3) and phosphorylated Ribosomal protein S6 (p-RPS6 (S235/236)) in BI-D1870-treated HL60 cells. Cell extracts were prepared at the indicated times after BI-D1870 treatment. β-Actin was used as an internal control. ( C ) Accumulation of metaphase cells following the treatment of BI-D1870. After treatment with BI-D1870 (5 μM) for the indicated times, HL60 cells were fixed and stained with DAPI and antibodies. Mitotic phases were further characterized in p-H3-positive populations by measuring the levels of Cyclin A and Cyclin B. Data represent the percentages of cell populations residing at each mitotic phase analyzed by the levels of Cyclin A and Cyclin B in the mitotic population. ( D ) Induction of mitotic arrest in KG1 cells by BI-D1870 treatment. KG1 cells were treated with BI-D1870 (5 μM) for indicated times. Cells were fixed and stained with DAPI and antibodies against p-H3, Cyclin A, and Cyclin B. Cell cycle profiles of DMSO or compound-treated KG1 cells were shown as the bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (top). Each mitotic phase distribution was identified as the cellular expression of Cyclin A and Cyclin B in mitotic cells (bottom). The percentage cell population at each cell cycle stage is shown. Flow cytometric profiles represent one out of three experiments with similar results. Data are graphed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.
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    ( A ) Cell cycle profiles of control and BI-D1870-treated HL60 cells by flow cytometry. HL60 cells were treated with BI-D1870 (5 μM). Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and antibodies. Bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (p-H3) was detected by multi-parameter flow cytometry. Percentages of cell populations at each cell cycle phase determined by DNA content (DAPI) and levels of p-H3, Cyclin A and Cyclin B were graphed. ( B ) Protein levels of G2/M phase markers (p-Rb (S780), <t>p-CDC2</t> <t>(Y15),</t> Cyclin B and Cyclin A), apoptosis marker (Cleaved Caspase 3) and phosphorylated Ribosomal protein S6 (p-RPS6 (S235/236)) in BI-D1870-treated HL60 cells. Cell extracts were prepared at the indicated times after BI-D1870 treatment. β-Actin was used as an internal control. ( C ) Accumulation of metaphase cells following the treatment of BI-D1870. After treatment with BI-D1870 (5 μM) for the indicated times, HL60 cells were fixed and stained with DAPI and antibodies. Mitotic phases were further characterized in p-H3-positive populations by measuring the levels of Cyclin A and Cyclin B. Data represent the percentages of cell populations residing at each mitotic phase analyzed by the levels of Cyclin A and Cyclin B in the mitotic population. ( D ) Induction of mitotic arrest in KG1 cells by BI-D1870 treatment. KG1 cells were treated with BI-D1870 (5 μM) for indicated times. Cells were fixed and stained with DAPI and antibodies against p-H3, Cyclin A, and Cyclin B. Cell cycle profiles of DMSO or compound-treated KG1 cells were shown as the bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (top). Each mitotic phase distribution was identified as the cellular expression of Cyclin A and Cyclin B in mitotic cells (bottom). The percentage cell population at each cell cycle stage is shown. Flow cytometric profiles represent one out of three experiments with similar results. Data are graphed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.
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    (A) Visualization of genomic DNA oxidized in wild-type (WT), TIA1 KO and TIAR KO MEFs by indirect immunofluorescence using an anti-8-oxo-dG antibody. WT MEFs were treated with 100 µM H 2 O 2 for 20 min to promote genomic DNA oxidation as a positive control. Scale bars represent 15 µm. (B) Analysis of cell-cycle phases by flow cytometry after propidium iodide staining. The data are means + SEM (n = 10; * P <0.05; ** P <0.01; *** P <0.001). (C) MEF cells knocked for TIA proteins showed delayed entry into G1/S. The above MEF cells were synchronized at G0/G1 by serum deprivation for 48–96 h. Samples were taken at 0, 16, 18 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (D) MEF cells knocked for TIA proteins showed delayed entry into S. The above MEF cells were synchronized at G1/S by Hydroxyurea blockage for 16–24 h and then released. Samples were taken at 0, 19, 22 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (E) MEF cells knocked for TIA proteins showed delayed entry into G0/G1. The above MEF cells were synchronized at G2/M by Nocodazole blockade for 24–30 h and then released. Samples were taken at 0, 3, 6, 17 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (F) Examples of MEF cells stained with To-Pro-3 (illustrated in green) and phalloidin-TRITC showing impairment of cytokinesis events in TIA1 and TIAR KO MEF. Scale bars represent 20 µm. (G) Analysis of the G2/M DNA damage checkpoint. Immunoblot of total (T), nuclear (N) and cytoplasmic (C) fractions (8 µg) from wild-type (WT), TIA1 KO and TIAR KO MEFs. The blot was probed with antibodies against U2AF65, α-tubulin, Cdc2 (total), Cdc2-P (Y15) and cyclin B1 proteins, as indicated. Molecular weight markers and the identities of protein bands are shown.

    Journal: PLoS ONE

    Article Title: T-cell Intracellular Antigen (TIA)-Proteins Deficiency in Murine Embryonic Fibroblasts Alters Cell Cycle Progression and Induces Autophagy

    doi: 10.1371/journal.pone.0075127

    Figure Lengend Snippet: (A) Visualization of genomic DNA oxidized in wild-type (WT), TIA1 KO and TIAR KO MEFs by indirect immunofluorescence using an anti-8-oxo-dG antibody. WT MEFs were treated with 100 µM H 2 O 2 for 20 min to promote genomic DNA oxidation as a positive control. Scale bars represent 15 µm. (B) Analysis of cell-cycle phases by flow cytometry after propidium iodide staining. The data are means + SEM (n = 10; * P <0.05; ** P <0.01; *** P <0.001). (C) MEF cells knocked for TIA proteins showed delayed entry into G1/S. The above MEF cells were synchronized at G0/G1 by serum deprivation for 48–96 h. Samples were taken at 0, 16, 18 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (D) MEF cells knocked for TIA proteins showed delayed entry into S. The above MEF cells were synchronized at G1/S by Hydroxyurea blockage for 16–24 h and then released. Samples were taken at 0, 19, 22 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (E) MEF cells knocked for TIA proteins showed delayed entry into G0/G1. The above MEF cells were synchronized at G2/M by Nocodazole blockade for 24–30 h and then released. Samples were taken at 0, 3, 6, 17 and 24 h after release, and the DNA content was measured by propidium iodide staining and FACS analysis. (F) Examples of MEF cells stained with To-Pro-3 (illustrated in green) and phalloidin-TRITC showing impairment of cytokinesis events in TIA1 and TIAR KO MEF. Scale bars represent 20 µm. (G) Analysis of the G2/M DNA damage checkpoint. Immunoblot of total (T), nuclear (N) and cytoplasmic (C) fractions (8 µg) from wild-type (WT), TIA1 KO and TIAR KO MEFs. The blot was probed with antibodies against U2AF65, α-tubulin, Cdc2 (total), Cdc2-P (Y15) and cyclin B1 proteins, as indicated. Molecular weight markers and the identities of protein bands are shown.

    Article Snippet: Immunoblots were carried out using the following antibodies: anti-TIA1 and anti-TIAR (Santa Cruz Biotechnology), anti-α-tubulin (Sigma), anti-U2AF65 (kindly provided by J. Valcárcel), anti-Cdc-2 and anti-Cdc2-P (Y15) (Cell Signaling), anti-Cyclin B1 (BD Pharmingen), anti-LC3B (Sigma), anti-p62 (Sigma) and anti-LAMP1 (DSHB).

    Techniques: Immunofluorescence, Positive Control, Flow Cytometry, Staining, Western Blot, Molecular Weight

    Targeting AKT3 and WEE1 increased p53 while reducing FOXM1 and CDK1 signaling. (A) and (B). Knockdown of WEE1 kinase led to a dose-dependent decrease in the phosphorylation of its substrate CDK1. Dose-dependent increase in the phosphorylation of H2AX was observed. Consequently, this resulted in increased p53, p21 as well as p27 levels, which are known to be inhibitory to cell proliferation. ERK2 served as a control for equal protein loading.

    Journal: Cancer Biology & Therapy

    Article Title: Identification of WEE1 as a target to make AKT inhibition more effective in melanoma

    doi: 10.1080/15384047.2017.1360446

    Figure Lengend Snippet: Targeting AKT3 and WEE1 increased p53 while reducing FOXM1 and CDK1 signaling. (A) and (B). Knockdown of WEE1 kinase led to a dose-dependent decrease in the phosphorylation of its substrate CDK1. Dose-dependent increase in the phosphorylation of H2AX was observed. Consequently, this resulted in increased p53, p21 as well as p27 levels, which are known to be inhibitory to cell proliferation. ERK2 served as a control for equal protein loading.

    Article Snippet: Primary antibodies used: p21 (sc-756), p27 (sc-528), p53 (sc-6243) and ERK2 (sc-1647) from Santa Cruz Biotechnology (Dallas, TX), pAKT (9271), AKT3 (3788), Total AKT (4685), pWEE1 (4910), WEE1 (4936), p-CDC2 (Y15) (9111), pRB (S807/811) (9308), pRB (S795) (9301), pRB (S780) (9307), (9309) and p-Histone H2AX (S139) (2577) from Cell Signaling (Danvers, MA).

    Techniques:

    Diagram showing the mechanism of synergism for co-targeting AKT and WEE1 signaling pathways. Inhibition of siWEE1 (1) suppresses inhibitory phosphorylation of CDK1 leading to early-G2/M progression. This leads DNA damage (2) and activates p53 signaling. p53 inhibits cell cycle progression by induction of p21, allowing DNA damage repair. If the DNA damage is not repairable, p53 induces apoptosis. However, in many cancer cells, apoptotic cascades are suppressed by oncogenic alterations. Over-activated AKT inhibits pro-apoptotic factors while inducing antiapoptotic factors (3). AKT signaling also enhances cell cycle progression by CyclinD1 mediated phosphorylation of RB (4) and inhibition of p27 (5). Furthermore, AKT phosphorylates and induces Polo-like kinase 1 (PLK1) (6), which in turn inhibits pro-apoptotic functions of p53 and its family members, p63 and p73 (7). In addition, PLK1 also induces FOXM1 activity and M-phase progression (8). Proteins that were validated by Western blotting are shown in bold.

    Journal: Cancer Biology & Therapy

    Article Title: Identification of WEE1 as a target to make AKT inhibition more effective in melanoma

    doi: 10.1080/15384047.2017.1360446

    Figure Lengend Snippet: Diagram showing the mechanism of synergism for co-targeting AKT and WEE1 signaling pathways. Inhibition of siWEE1 (1) suppresses inhibitory phosphorylation of CDK1 leading to early-G2/M progression. This leads DNA damage (2) and activates p53 signaling. p53 inhibits cell cycle progression by induction of p21, allowing DNA damage repair. If the DNA damage is not repairable, p53 induces apoptosis. However, in many cancer cells, apoptotic cascades are suppressed by oncogenic alterations. Over-activated AKT inhibits pro-apoptotic factors while inducing antiapoptotic factors (3). AKT signaling also enhances cell cycle progression by CyclinD1 mediated phosphorylation of RB (4) and inhibition of p27 (5). Furthermore, AKT phosphorylates and induces Polo-like kinase 1 (PLK1) (6), which in turn inhibits pro-apoptotic functions of p53 and its family members, p63 and p73 (7). In addition, PLK1 also induces FOXM1 activity and M-phase progression (8). Proteins that were validated by Western blotting are shown in bold.

    Article Snippet: Primary antibodies used: p21 (sc-756), p27 (sc-528), p53 (sc-6243) and ERK2 (sc-1647) from Santa Cruz Biotechnology (Dallas, TX), pAKT (9271), AKT3 (3788), Total AKT (4685), pWEE1 (4910), WEE1 (4936), p-CDC2 (Y15) (9111), pRB (S807/811) (9308), pRB (S795) (9301), pRB (S780) (9307), (9309) and p-Histone H2AX (S139) (2577) from Cell Signaling (Danvers, MA).

    Techniques: Inhibition, Activity Assay, Western Blot

    Depletion of slp1 arrests cells in metaphase but cannot prevent cytokinesis. (A) Overview of the experimental setup used to repress the expression of slp1 under the control of p41-nmt1 . Slp1 is repressed in the presence of 15 µM thiamine (+T) and expressed in the absence of thiamine (−T). Wild type cells were used as control for the endogenous expression of slp1. (B,D–F) cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for mRNA expression, microscopy, and western blot. (B) mRNA expression of slp1 in wild type and p41-nmt1:slp1 cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of four biological replicates are shown. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test. ns: p > 0.05; ****: p ≤ 0.0001. (C) Image of wild type andp41- nmt1:slp1 cells streaked on EMM agar plates that contain 15 µM thiamine or not at 30°C. (D) Images of ethanol-fixed cells stained with DAPI to visualise DNA compactation and blankophor to visualise cell wall and septum by microscopy. In the lower panels, Differently-colored arrowheads indicate the different phenotypes scored in (E) : Cycling cells (red), metaphase-arrested cells with condensed chromosomes (cyan), cut cells (purple) and septated cells with unequal segregation of chromosomes (yellow). Scale bar 5 µm. (E) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (F) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 served as indicators of CDK activity and were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast

    doi: 10.3389/fcell.2022.876810

    Figure Lengend Snippet: Depletion of slp1 arrests cells in metaphase but cannot prevent cytokinesis. (A) Overview of the experimental setup used to repress the expression of slp1 under the control of p41-nmt1 . Slp1 is repressed in the presence of 15 µM thiamine (+T) and expressed in the absence of thiamine (−T). Wild type cells were used as control for the endogenous expression of slp1. (B,D–F) cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for mRNA expression, microscopy, and western blot. (B) mRNA expression of slp1 in wild type and p41-nmt1:slp1 cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of four biological replicates are shown. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test. ns: p > 0.05; ****: p ≤ 0.0001. (C) Image of wild type andp41- nmt1:slp1 cells streaked on EMM agar plates that contain 15 µM thiamine or not at 30°C. (D) Images of ethanol-fixed cells stained with DAPI to visualise DNA compactation and blankophor to visualise cell wall and septum by microscopy. In the lower panels, Differently-colored arrowheads indicate the different phenotypes scored in (E) : Cycling cells (red), metaphase-arrested cells with condensed chromosomes (cyan), cut cells (purple) and septated cells with unequal segregation of chromosomes (yellow). Scale bar 5 µm. (E) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (F) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 served as indicators of CDK activity and were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2.

    Article Snippet: The following primary antibodies were used in this study: mouse anti-phospho-(Thr-Pro-101) to detect Cdk substrate-specific phosphorylations (1:1,000, Cell Signalling Technology), rabbit anti-phospho-cdc2 (Tyr15) to detect Cdc2-Y15-P (1:1,000, Cell Signalling Technology), anti-Cdc13 antibody (6F11/2) (1:1,000, Abcam), anti-Cdc2 (PSTAIR) (1:1,000, Millipore Sigma), anti-Byr4 (a kind gift from Prof. Kathleen Gould, 1:5,000).

    Techniques: Expressing, Microscopy, Western Blot, Staining, Activity Assay

    Protein phosphatases influence the mitotic arrest. (A–F) cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A,B) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Y15-Cdc2 phosphorylation in p41-nmt1:slp1 and p41-nmt1:slp1 clp1Δ (A) or p41-nmt1:slp1 and p41-nmt1:slp1 ppa2Δ strains (B) were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (C,D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E,F) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C,D) . Scale bar 5 µm.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast

    doi: 10.3389/fcell.2022.876810

    Figure Lengend Snippet: Protein phosphatases influence the mitotic arrest. (A–F) cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A,B) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Y15-Cdc2 phosphorylation in p41-nmt1:slp1 and p41-nmt1:slp1 clp1Δ (A) or p41-nmt1:slp1 and p41-nmt1:slp1 ppa2Δ strains (B) were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (C,D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E,F) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C,D) . Scale bar 5 µm.

    Article Snippet: The following primary antibodies were used in this study: mouse anti-phospho-(Thr-Pro-101) to detect Cdk substrate-specific phosphorylations (1:1,000, Cell Signalling Technology), rabbit anti-phospho-cdc2 (Tyr15) to detect Cdc2-Y15-P (1:1,000, Cell Signalling Technology), anti-Cdc13 antibody (6F11/2) (1:1,000, Abcam), anti-Cdc2 (PSTAIR) (1:1,000, Millipore Sigma), anti-Byr4 (a kind gift from Prof. Kathleen Gould, 1:5,000).

    Techniques: Microscopy, Western Blot, Staining

    B55 Pab1 and B56 Par1 PP2A-regulatory subunits have opposite effects in the induction of septation in metaphase arrested cells. (A–C) The indicated strains were grown in EMM and then shifted to EMM with 0.5 mM 1-Naphthaleneacetic acid (auxin) for Pab1 degradation and/or 15 µm thiamine ( 41nmt promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A) Depletion of Pab1 was detected by western blot against its N-terminal 3PK tag. See also . Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 served as indicators of CDK activity and were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (B) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (C) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells of p41-nmt1:slp1p41-nmt1-3PK-miniAID-pab1 in (B) . Scale bar 5 µm. (D–F) p41 - nmt1:slp1 par1Δ cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells of p41-nmt1:slp1 par1Δ in (D) . Scale bar 5 µm. (F) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 in p41-nmt1:slp1 and p41-nmt1:slp1 par1Δ strains were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast

    doi: 10.3389/fcell.2022.876810

    Figure Lengend Snippet: B55 Pab1 and B56 Par1 PP2A-regulatory subunits have opposite effects in the induction of septation in metaphase arrested cells. (A–C) The indicated strains were grown in EMM and then shifted to EMM with 0.5 mM 1-Naphthaleneacetic acid (auxin) for Pab1 degradation and/or 15 µm thiamine ( 41nmt promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A) Depletion of Pab1 was detected by western blot against its N-terminal 3PK tag. See also . Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 served as indicators of CDK activity and were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (B) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (C) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells of p41-nmt1:slp1p41-nmt1-3PK-miniAID-pab1 in (B) . Scale bar 5 µm. (D–F) p41 - nmt1:slp1 par1Δ cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells of p41-nmt1:slp1 par1Δ in (D) . Scale bar 5 µm. (F) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 in p41-nmt1:slp1 and p41-nmt1:slp1 par1Δ strains were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2.

    Article Snippet: The following primary antibodies were used in this study: mouse anti-phospho-(Thr-Pro-101) to detect Cdk substrate-specific phosphorylations (1:1,000, Cell Signalling Technology), rabbit anti-phospho-cdc2 (Tyr15) to detect Cdc2-Y15-P (1:1,000, Cell Signalling Technology), anti-Cdc13 antibody (6F11/2) (1:1,000, Abcam), anti-Cdc2 (PSTAIR) (1:1,000, Millipore Sigma), anti-Byr4 (a kind gift from Prof. Kathleen Gould, 1:5,000).

    Techniques: Microscopy, Western Blot, Activity Assay, Staining

    Interplay between CDK and PP2A-B55 Pab1 in the regulation of septation. (A–F) Cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A,B) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 in p41-nmt1:slp1 , p41-nmt1:slp1 par1Δ and p41-nmt1:slp1 cdc2-3w par1Δ (A) or p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ strains (B) were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (C,D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) in p41-nmt1:slp1 cdc2-3w par1Δ (C) and p41-nmt1:slp1 igo1Δ (D) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E,F) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C,D) . Scale bar 5 µm.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast

    doi: 10.3389/fcell.2022.876810

    Figure Lengend Snippet: Interplay between CDK and PP2A-B55 Pab1 in the regulation of septation. (A–F) Cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. (A,B) Cdc13 levels, phosphorylation at T/P sites in CDK substrates and Cdc2 phosphorylated at Y15 in p41-nmt1:slp1 , p41-nmt1:slp1 par1Δ and p41-nmt1:slp1 cdc2-3w par1Δ (A) or p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ strains (B) were detected by western blot. Cdc2 (PSTAIR) was used as a protein loading control. The numbers under the western blot indicate CDK-substrate Phosphorylation (P-T/P) quantification relative to Cdc2. (C,D) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) in p41-nmt1:slp1 cdc2-3w par1Δ (C) and p41-nmt1:slp1 igo1Δ (D) were determined at the indicated time points after the addition of thiamine to the culture. At least 200 cells were counted per time point. (E,F) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C,D) . Scale bar 5 µm.

    Article Snippet: The following primary antibodies were used in this study: mouse anti-phospho-(Thr-Pro-101) to detect Cdk substrate-specific phosphorylations (1:1,000, Cell Signalling Technology), rabbit anti-phospho-cdc2 (Tyr15) to detect Cdc2-Y15-P (1:1,000, Cell Signalling Technology), anti-Cdc13 antibody (6F11/2) (1:1,000, Abcam), anti-Cdc2 (PSTAIR) (1:1,000, Millipore Sigma), anti-Byr4 (a kind gift from Prof. Kathleen Gould, 1:5,000).

    Techniques: Microscopy, Western Blot, Staining

    Dynamic Byr4 phosphorylation and Cdc13 localisation during a sustained metaphase arrest. (A–D) Cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. For the nda3-3KM311 mutant, cells were grown at 30 and then shifted to 18°C for 6 h. (A,C) Byr4 levels and phosphorylation (band shift) were detected by western blot in p41-nmt1:slp1 , p41-nmt1:slp1 ppa2Δ , p41-nmt1:slp1 par1Δ and nda3-3KM311 mutant (A) ; p41-nmt1:slp1 , p41-nmt1:slp1 par1Δ and p41-nmt1:slp1 cdc2-3w par1Δ (B) and p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ (B) or p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ strains (C) in EMM -T and EMM +T at the indicated time points. Cdc2 (PSTAIR) was used as a protein loading control. (D) Representative images of cdc13mCherry localization and Blankophor-stained ethanol-fixed cells upon slp1 repression by thiamine. Scale bar 5 µm.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast

    doi: 10.3389/fcell.2022.876810

    Figure Lengend Snippet: Dynamic Byr4 phosphorylation and Cdc13 localisation during a sustained metaphase arrest. (A–D) Cells were grown exponentially in EMM ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C. Samples were collected at the indicated time points for microscopy and western blot. For the nda3-3KM311 mutant, cells were grown at 30 and then shifted to 18°C for 6 h. (A,C) Byr4 levels and phosphorylation (band shift) were detected by western blot in p41-nmt1:slp1 , p41-nmt1:slp1 ppa2Δ , p41-nmt1:slp1 par1Δ and nda3-3KM311 mutant (A) ; p41-nmt1:slp1 , p41-nmt1:slp1 par1Δ and p41-nmt1:slp1 cdc2-3w par1Δ (B) and p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ (B) or p41-nmt1:slp1 and p41-nmt1:slp1 igo1Δ strains (C) in EMM -T and EMM +T at the indicated time points. Cdc2 (PSTAIR) was used as a protein loading control. (D) Representative images of cdc13mCherry localization and Blankophor-stained ethanol-fixed cells upon slp1 repression by thiamine. Scale bar 5 µm.

    Article Snippet: The following primary antibodies were used in this study: mouse anti-phospho-(Thr-Pro-101) to detect Cdk substrate-specific phosphorylations (1:1,000, Cell Signalling Technology), rabbit anti-phospho-cdc2 (Tyr15) to detect Cdc2-Y15-P (1:1,000, Cell Signalling Technology), anti-Cdc13 antibody (6F11/2) (1:1,000, Abcam), anti-Cdc2 (PSTAIR) (1:1,000, Millipore Sigma), anti-Byr4 (a kind gift from Prof. Kathleen Gould, 1:5,000).

    Techniques: Microscopy, Western Blot, Mutagenesis, Electrophoretic Mobility Shift Assay, Staining

    The mitotic transcriptional wave influences the behaviour of cells arrested in metaphase. (A–D) Cells were grown exponentially in EMM at 30°C or 25°C ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C or 36°C. Samples were collected at the indicated time points for mRNA expression and microscopy. (A) mRNA expression of slp1, ace2, gas1 and par2 in wild type and in p41-nmt1:slp1 or p41-nmt1:slp1 par1Δ cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of three biological replicate are shown. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test. ns: p > 0.05; *: p ≤ 0.05; ** ≤ 0.01; ****: p ≤ 0.0001. (B) Left panel. mRNA expression of ace2 in p41-nmt1:slp1 or p41-nmt1:slp1 par1Δ , p41-nmt1:slp1 cdc2-3w par1Δ and p41-nmt1:slp1 igo1Δ cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of at least two biological replicates are shown. Right panel. mRNA expression of ace2 in p41-nmt1:slp1 or p41-nmt1:slp1 sak1ts cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of at least two biological replicates are shown. (C) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) in p41-nmt1:slp1 (left panel) and p41-nmt1:slp1 sak1ts (right panel) at each indicated time point after thiamine addition. (*) cells grown at 25°C. At least 200 cells were counted per time point. (D) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C) . Scale bar 5 µm.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast

    doi: 10.3389/fcell.2022.876810

    Figure Lengend Snippet: The mitotic transcriptional wave influences the behaviour of cells arrested in metaphase. (A–D) Cells were grown exponentially in EMM at 30°C or 25°C ( p41-nmt1 promoter induced) and shifted to EMM +T ( p41-nmt1 promoter repressed) at 30°C or 36°C. Samples were collected at the indicated time points for mRNA expression and microscopy. (A) mRNA expression of slp1, ace2, gas1 and par2 in wild type and in p41-nmt1:slp1 or p41-nmt1:slp1 par1Δ cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of three biological replicate are shown. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test. ns: p > 0.05; *: p ≤ 0.05; ** ≤ 0.01; ****: p ≤ 0.0001. (B) Left panel. mRNA expression of ace2 in p41-nmt1:slp1 or p41-nmt1:slp1 par1Δ , p41-nmt1:slp1 cdc2-3w par1Δ and p41-nmt1:slp1 igo1Δ cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of at least two biological replicates are shown. Right panel. mRNA expression of ace2 in p41-nmt1:slp1 or p41-nmt1:slp1 sak1ts cells. Expression is relative to actin and was determined by qPCR. Mean and SEM of at least two biological replicates are shown. (C) The percentage of cycling cells, metaphase-arrested cells, and arrested cells that underwent cytokinesis (cut or unequal) in p41-nmt1:slp1 (left panel) and p41-nmt1:slp1 sak1ts (right panel) at each indicated time point after thiamine addition. (*) cells grown at 25°C. At least 200 cells were counted per time point. (D) Representative images of mitotic features distributions from DAPI and Blankophor-stained ethanol-fixed cells in (C) . Scale bar 5 µm.

    Article Snippet: The following primary antibodies were used in this study: mouse anti-phospho-(Thr-Pro-101) to detect Cdk substrate-specific phosphorylations (1:1,000, Cell Signalling Technology), rabbit anti-phospho-cdc2 (Tyr15) to detect Cdc2-Y15-P (1:1,000, Cell Signalling Technology), anti-Cdc13 antibody (6F11/2) (1:1,000, Abcam), anti-Cdc2 (PSTAIR) (1:1,000, Millipore Sigma), anti-Byr4 (a kind gift from Prof. Kathleen Gould, 1:5,000).

    Techniques: Expressing, Microscopy, Staining

    Proposed model for the regulation of cytokinesis by PP2A phosphatases and mitotic CDK complex during a prolonged mitotic arrest. (A) Mitotic arrest was induced through transcriptional repression of the APC/C activator Slp1 (under the control of the thiamine-repressible promoter p41-nmt1 ). Silencing of slp1 resulted in the rapid accumulation of the mitotic cyclin Cdc13, increased mitotic phosphorylation and chromosome condensation. Upon prolonged periods of mitotic arrest, mitotic dephosphorylation and induction of cytokinesis could be observed in the presence of condensed chromosomes and high levels of Cdc13. (B) Mitotic CDK both favors and represses cytokinesis by inactivating the Spg1 GAP (activation of the top of the SIN cascade), while preventing recruitment of Sid1:Cdc14 (repression of the bottom of the SIN cascade) ( ; ; ). PP2A-B55 Pab1 and PP2A-B56 Par1 have opposite effects in the regulation of cytokinesis: PP2A-B55 Pab1 represses cytokinesis and, in its absence, untimely septation in the presence of unsegregated chromosomes occurs at high frequency in mitotically-arrested cells (1). The mitotic CDK complex establishes a double negative feedback loop with PP2A-B55 Pab1 through the Ppk18-Igo1 (Greatwall-ENSA) pathway (2). In consequence, high CDK activity during a prolonged mitotic arrest would result in PP2A-B55 Pab1 inactivation and induction of cytokinesis. In our experimental conditions, engagement of this double negative feedback loop would be sufficient to overcome the repression of cytokinesis by CDK. PP2A-B56 Par1 favors cytokinesis through different means: it participates in the activation of the mitotic CDK complex at the G2/M transition (3). In its absence, cells enter mitosis with residual Cdc2-Tyr15 phosphorylation. Presumably, this hinders the repression of PP2A-B55 Pab1 by CDK and results in septation being prevented. The underlying mechanism of this regulation is unknown at present, but might involve modulation of Wee1 and/or Cdc25 activities. In addition, PP2A-B56 Par1 participates in the activation of the mitotic transcriptional program brought about by the RFX transcription factor Sak1 (4). Loss of either Sak1 or PP2A-B56 Par1 impairs the expression of cytokinetic regulators and this contributes to the absence of cytokinesis during the arrest in these mutants. (C) Steady mitotic dephosphorylation during the arrest requires the activities of Clp1, PP2A-B55 Pab1 and PP2A-B56 Par1 .

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast

    doi: 10.3389/fcell.2022.876810

    Figure Lengend Snippet: Proposed model for the regulation of cytokinesis by PP2A phosphatases and mitotic CDK complex during a prolonged mitotic arrest. (A) Mitotic arrest was induced through transcriptional repression of the APC/C activator Slp1 (under the control of the thiamine-repressible promoter p41-nmt1 ). Silencing of slp1 resulted in the rapid accumulation of the mitotic cyclin Cdc13, increased mitotic phosphorylation and chromosome condensation. Upon prolonged periods of mitotic arrest, mitotic dephosphorylation and induction of cytokinesis could be observed in the presence of condensed chromosomes and high levels of Cdc13. (B) Mitotic CDK both favors and represses cytokinesis by inactivating the Spg1 GAP (activation of the top of the SIN cascade), while preventing recruitment of Sid1:Cdc14 (repression of the bottom of the SIN cascade) ( ; ; ). PP2A-B55 Pab1 and PP2A-B56 Par1 have opposite effects in the regulation of cytokinesis: PP2A-B55 Pab1 represses cytokinesis and, in its absence, untimely septation in the presence of unsegregated chromosomes occurs at high frequency in mitotically-arrested cells (1). The mitotic CDK complex establishes a double negative feedback loop with PP2A-B55 Pab1 through the Ppk18-Igo1 (Greatwall-ENSA) pathway (2). In consequence, high CDK activity during a prolonged mitotic arrest would result in PP2A-B55 Pab1 inactivation and induction of cytokinesis. In our experimental conditions, engagement of this double negative feedback loop would be sufficient to overcome the repression of cytokinesis by CDK. PP2A-B56 Par1 favors cytokinesis through different means: it participates in the activation of the mitotic CDK complex at the G2/M transition (3). In its absence, cells enter mitosis with residual Cdc2-Tyr15 phosphorylation. Presumably, this hinders the repression of PP2A-B55 Pab1 by CDK and results in septation being prevented. The underlying mechanism of this regulation is unknown at present, but might involve modulation of Wee1 and/or Cdc25 activities. In addition, PP2A-B56 Par1 participates in the activation of the mitotic transcriptional program brought about by the RFX transcription factor Sak1 (4). Loss of either Sak1 or PP2A-B56 Par1 impairs the expression of cytokinetic regulators and this contributes to the absence of cytokinesis during the arrest in these mutants. (C) Steady mitotic dephosphorylation during the arrest requires the activities of Clp1, PP2A-B55 Pab1 and PP2A-B56 Par1 .

    Article Snippet: The following primary antibodies were used in this study: mouse anti-phospho-(Thr-Pro-101) to detect Cdk substrate-specific phosphorylations (1:1,000, Cell Signalling Technology), rabbit anti-phospho-cdc2 (Tyr15) to detect Cdc2-Y15-P (1:1,000, Cell Signalling Technology), anti-Cdc13 antibody (6F11/2) (1:1,000, Abcam), anti-Cdc2 (PSTAIR) (1:1,000, Millipore Sigma), anti-Byr4 (a kind gift from Prof. Kathleen Gould, 1:5,000).

    Techniques: De-Phosphorylation Assay, Activation Assay, Activity Assay, Expressing

    ( A ) Cell cycle profiles of control and BI-D1870-treated HL60 cells by flow cytometry. HL60 cells were treated with BI-D1870 (5 μM). Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and antibodies. Bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (p-H3) was detected by multi-parameter flow cytometry. Percentages of cell populations at each cell cycle phase determined by DNA content (DAPI) and levels of p-H3, Cyclin A and Cyclin B were graphed. ( B ) Protein levels of G2/M phase markers (p-Rb (S780), p-CDC2 (Y15), Cyclin B and Cyclin A), apoptosis marker (Cleaved Caspase 3) and phosphorylated Ribosomal protein S6 (p-RPS6 (S235/236)) in BI-D1870-treated HL60 cells. Cell extracts were prepared at the indicated times after BI-D1870 treatment. β-Actin was used as an internal control. ( C ) Accumulation of metaphase cells following the treatment of BI-D1870. After treatment with BI-D1870 (5 μM) for the indicated times, HL60 cells were fixed and stained with DAPI and antibodies. Mitotic phases were further characterized in p-H3-positive populations by measuring the levels of Cyclin A and Cyclin B. Data represent the percentages of cell populations residing at each mitotic phase analyzed by the levels of Cyclin A and Cyclin B in the mitotic population. ( D ) Induction of mitotic arrest in KG1 cells by BI-D1870 treatment. KG1 cells were treated with BI-D1870 (5 μM) for indicated times. Cells were fixed and stained with DAPI and antibodies against p-H3, Cyclin A, and Cyclin B. Cell cycle profiles of DMSO or compound-treated KG1 cells were shown as the bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (top). Each mitotic phase distribution was identified as the cellular expression of Cyclin A and Cyclin B in mitotic cells (bottom). The percentage cell population at each cell cycle stage is shown. Flow cytometric profiles represent one out of three experiments with similar results. Data are graphed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.

    Journal: Oncotarget

    Article Title: RSK inhibitor BI-D1870 inhibits acute myeloid leukemia cell proliferation by targeting mitotic exit

    doi: 10.18632/oncotarget.27630

    Figure Lengend Snippet: ( A ) Cell cycle profiles of control and BI-D1870-treated HL60 cells by flow cytometry. HL60 cells were treated with BI-D1870 (5 μM). Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and antibodies. Bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (p-H3) was detected by multi-parameter flow cytometry. Percentages of cell populations at each cell cycle phase determined by DNA content (DAPI) and levels of p-H3, Cyclin A and Cyclin B were graphed. ( B ) Protein levels of G2/M phase markers (p-Rb (S780), p-CDC2 (Y15), Cyclin B and Cyclin A), apoptosis marker (Cleaved Caspase 3) and phosphorylated Ribosomal protein S6 (p-RPS6 (S235/236)) in BI-D1870-treated HL60 cells. Cell extracts were prepared at the indicated times after BI-D1870 treatment. β-Actin was used as an internal control. ( C ) Accumulation of metaphase cells following the treatment of BI-D1870. After treatment with BI-D1870 (5 μM) for the indicated times, HL60 cells were fixed and stained with DAPI and antibodies. Mitotic phases were further characterized in p-H3-positive populations by measuring the levels of Cyclin A and Cyclin B. Data represent the percentages of cell populations residing at each mitotic phase analyzed by the levels of Cyclin A and Cyclin B in the mitotic population. ( D ) Induction of mitotic arrest in KG1 cells by BI-D1870 treatment. KG1 cells were treated with BI-D1870 (5 μM) for indicated times. Cells were fixed and stained with DAPI and antibodies against p-H3, Cyclin A, and Cyclin B. Cell cycle profiles of DMSO or compound-treated KG1 cells were shown as the bivariate distribution of DNA content versus the level of phosphorylated Histone H3 (top). Each mitotic phase distribution was identified as the cellular expression of Cyclin A and Cyclin B in mitotic cells (bottom). The percentage cell population at each cell cycle stage is shown. Flow cytometric profiles represent one out of three experiments with similar results. Data are graphed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.

    Article Snippet: The following antibodies were used in intracellular protein staining: FITC-conjugated anti-Cyclin A (clone BF683), PE-conjugated anti-Cyclin B (clone GNS-1), Alexa Fluor 647 or Alexa Fluor 700-conjugated anti-cPARP (clone F21-852, BD Biosciences); PECy7-conjugated ant-pH3 (pS28) (clone HTA283, Biolegend); anti-Securin (EPR3240, Abcam, Cambridge, MA); anti-p-CDC2 (Y15) (clone 10A11, Cell Signaling Technology, Danvers, MA, USA); Alexa Fluor 647-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA).

    Techniques: Flow Cytometry, Staining, Marker, Expressing

    HL60 cells were treated with BI-D1870 (5 μM) for the indicated hours, and then cells were collected and analyzed for the cellular levels of p-CDC25C (S198) ( A ) or p-CDC2 (Y15) ( B ). Fixed cells were stained with DAPI and antibodies against Cyclin A, Cyclin B, p-H3, p-CDC2 (Y15), and p-CDC25C (S198). (A) Decrease in positive phosphorylation at serine 198 (S198) on CDC25C following the treatment of BI-D1870. Representative plot of p-CDC25C (S198) levels in G2 and mitotic phases populations following the treatment of BI-D1870. The graph shows the MFI of p-CDC25C (S198) in G2 and mitotic phases populations of HL60 cells treated with or without BI-D1870. (B) Temporary inhibition of CDC2 activation in the G2 phase by BI-D1870 treatment. CDC2 activity is regulated in a negative fashion by phosphorylation at tyrosine 15 (Y15). Representative flow cytometric profile of p-CDC2 (Y15) levels in the G2 and mitotic phases populations following the treatment of BI-D1870. The graph shows median fluorescence intensities (MFI) of p-CDC2 (Y15) in the G2 and mitotic phases populations of HL60 cells treated with or without BI-D1870. Negative phosphorylation at Y15 on CDC2 was transiently enhanced in only the G2 phase at 2 h after BI-D1870 treatment. Flow cytometric profiles represent one out of three independent experiments. Values are graphed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01.

    Journal: Oncotarget

    Article Title: RSK inhibitor BI-D1870 inhibits acute myeloid leukemia cell proliferation by targeting mitotic exit

    doi: 10.18632/oncotarget.27630

    Figure Lengend Snippet: HL60 cells were treated with BI-D1870 (5 μM) for the indicated hours, and then cells were collected and analyzed for the cellular levels of p-CDC25C (S198) ( A ) or p-CDC2 (Y15) ( B ). Fixed cells were stained with DAPI and antibodies against Cyclin A, Cyclin B, p-H3, p-CDC2 (Y15), and p-CDC25C (S198). (A) Decrease in positive phosphorylation at serine 198 (S198) on CDC25C following the treatment of BI-D1870. Representative plot of p-CDC25C (S198) levels in G2 and mitotic phases populations following the treatment of BI-D1870. The graph shows the MFI of p-CDC25C (S198) in G2 and mitotic phases populations of HL60 cells treated with or without BI-D1870. (B) Temporary inhibition of CDC2 activation in the G2 phase by BI-D1870 treatment. CDC2 activity is regulated in a negative fashion by phosphorylation at tyrosine 15 (Y15). Representative flow cytometric profile of p-CDC2 (Y15) levels in the G2 and mitotic phases populations following the treatment of BI-D1870. The graph shows median fluorescence intensities (MFI) of p-CDC2 (Y15) in the G2 and mitotic phases populations of HL60 cells treated with or without BI-D1870. Negative phosphorylation at Y15 on CDC2 was transiently enhanced in only the G2 phase at 2 h after BI-D1870 treatment. Flow cytometric profiles represent one out of three independent experiments. Values are graphed as mean ± SEM ( n = 3). * p < 0.05; ** p < 0.01.

    Article Snippet: The following antibodies were used in intracellular protein staining: FITC-conjugated anti-Cyclin A (clone BF683), PE-conjugated anti-Cyclin B (clone GNS-1), Alexa Fluor 647 or Alexa Fluor 700-conjugated anti-cPARP (clone F21-852, BD Biosciences); PECy7-conjugated ant-pH3 (pS28) (clone HTA283, Biolegend); anti-Securin (EPR3240, Abcam, Cambridge, MA); anti-p-CDC2 (Y15) (clone 10A11, Cell Signaling Technology, Danvers, MA, USA); Alexa Fluor 647-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA).

    Techniques: Staining, Inhibition, Activation Assay, Activity Assay, Fluorescence