rabbit total cdc2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit total cdc2
    A , A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of <t>CDC2</t> (pCDC2 Y15 ), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. B , PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 Y15 ), serine-216 phosphorylation of CDC25C (pCDC25c S216 ), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. C , drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. D , PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. E , A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 Y15 , and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.
    Rabbit Total Cdc2, 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/rabbit total cdc2/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells"

    Article Title: Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014335

    A , A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of CDC2 (pCDC2 Y15 ), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. B , PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 Y15 ), serine-216 phosphorylation of CDC25C (pCDC25c S216 ), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. C , drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. D , PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. E , A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 Y15 , and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.
    Figure Legend Snippet: A , A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of CDC2 (pCDC2 Y15 ), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. B , PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 Y15 ), serine-216 phosphorylation of CDC25C (pCDC25c S216 ), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. C , drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. D , PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. E , A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 Y15 , and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.

    Techniques Used: Cell Culture, Expressing, Western Blot, Software, Flow Cytometry

    A549 cells were untreated or treated with LBH589 (40 nM) for various time points. Cell lysates were prepared, and protein expression levels of cPARP, CHK1, Tyr15 phosphorylation of pCDC2 (pCDC2 Y15 ), Ser216 phosphorylation of CDC25C (pCDC25 S216 ), acetyl-H4, and cyclin B1 were determined. B , Quantitative Chk1 mRNA expression analysis. Total RNA was prepared from A549 cells after 24 hours of treatment with 40 nM LBH589 or vehicle. mRNA expression levels were quantified using Real-time PCR analysis. All results were normalized to GAPDH mRNA levels, and the mean and standard deviations values from four independent experiments are shown. C , Ectopic expression of Chk1 reverses HDACi-induced apoptosis but not histone acetylation. A549 cells were transiently transfected with an empty vector (control) or GFP- or FLAG-tagged (GFP-CHK1 or FLAG-CHK1) Chk1 expression plasmid. Forty-eight hours after transfection, cells were cultured without (control, C) or with LBH589 (L) (40 nM) for an additional 24 hour before harvesting for Western blot analysis. Treatment-induced changes in cPARP, acetyl-H4, phospho-CDC2 Y15 , and ectopically expressed GFP-CHK1 or FLAG-CHK1 proteins were determined by Western blot analysis. β-actin expression was used as loading control. Experiments were repeated 3 times, and a representative experiment is shown. The arrows show the position of the GFP-CHK1 and FLAG-CHK1 proteins. D , in primary NSCLC patient samples, Chk1 protein downregulation correlates with increased cPARP ex vivo . Tumor samples were collected with a 23-gauge needle from patient-derived tumors, and cells were treated in duplicate with vehicle (control) or LBH589 (40 nM) for 18 hours. Following treatment, adherent and non-adherent cells were pooled, cell extracts were prepared, and expression levels of cPARP, Chk1, and acetyl-H3 were analyzed by Western blot. β-actin expression was used as loading control. SCC: squamous cell carcinoma; AC: adenocarcinoma.
    Figure Legend Snippet: A549 cells were untreated or treated with LBH589 (40 nM) for various time points. Cell lysates were prepared, and protein expression levels of cPARP, CHK1, Tyr15 phosphorylation of pCDC2 (pCDC2 Y15 ), Ser216 phosphorylation of CDC25C (pCDC25 S216 ), acetyl-H4, and cyclin B1 were determined. B , Quantitative Chk1 mRNA expression analysis. Total RNA was prepared from A549 cells after 24 hours of treatment with 40 nM LBH589 or vehicle. mRNA expression levels were quantified using Real-time PCR analysis. All results were normalized to GAPDH mRNA levels, and the mean and standard deviations values from four independent experiments are shown. C , Ectopic expression of Chk1 reverses HDACi-induced apoptosis but not histone acetylation. A549 cells were transiently transfected with an empty vector (control) or GFP- or FLAG-tagged (GFP-CHK1 or FLAG-CHK1) Chk1 expression plasmid. Forty-eight hours after transfection, cells were cultured without (control, C) or with LBH589 (L) (40 nM) for an additional 24 hour before harvesting for Western blot analysis. Treatment-induced changes in cPARP, acetyl-H4, phospho-CDC2 Y15 , and ectopically expressed GFP-CHK1 or FLAG-CHK1 proteins were determined by Western blot analysis. β-actin expression was used as loading control. Experiments were repeated 3 times, and a representative experiment is shown. The arrows show the position of the GFP-CHK1 and FLAG-CHK1 proteins. D , in primary NSCLC patient samples, Chk1 protein downregulation correlates with increased cPARP ex vivo . Tumor samples were collected with a 23-gauge needle from patient-derived tumors, and cells were treated in duplicate with vehicle (control) or LBH589 (40 nM) for 18 hours. Following treatment, adherent and non-adherent cells were pooled, cell extracts were prepared, and expression levels of cPARP, Chk1, and acetyl-H3 were analyzed by Western blot. β-actin expression was used as loading control. SCC: squamous cell carcinoma; AC: adenocarcinoma.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Ex Vivo, Derivative Assay

    rabbit total cdc2  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
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    Structured Review

    Cell Signaling Technology Inc rabbit total cdc2
    A , A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of <t>CDC2</t> (pCDC2 Y15 ), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. B , PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 Y15 ), serine-216 phosphorylation of CDC25C (pCDC25c S216 ), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. C , drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. D , PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. E , A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 Y15 , and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.
    Rabbit Total Cdc2, 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/rabbit total cdc2/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit total cdc2 - by Bioz Stars, 2023-03
    96/100 stars

    Images

    1) Product Images from "Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells"

    Article Title: Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014335

    A , A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of CDC2 (pCDC2 Y15 ), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. B , PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 Y15 ), serine-216 phosphorylation of CDC25C (pCDC25c S216 ), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. C , drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. D , PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. E , A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 Y15 , and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.
    Figure Legend Snippet: A , A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of CDC2 (pCDC2 Y15 ), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. B , PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 Y15 ), serine-216 phosphorylation of CDC25C (pCDC25c S216 ), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. C , drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. D , PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. E , A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 Y15 , and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.

    Techniques Used: Cell Culture, Expressing, Western Blot, Software, Flow Cytometry

    A549 cells were untreated or treated with LBH589 (40 nM) for various time points. Cell lysates were prepared, and protein expression levels of cPARP, CHK1, Tyr15 phosphorylation of pCDC2 (pCDC2 Y15 ), Ser216 phosphorylation of CDC25C (pCDC25 S216 ), acetyl-H4, and cyclin B1 were determined. B , Quantitative Chk1 mRNA expression analysis. Total RNA was prepared from A549 cells after 24 hours of treatment with 40 nM LBH589 or vehicle. mRNA expression levels were quantified using Real-time PCR analysis. All results were normalized to GAPDH mRNA levels, and the mean and standard deviations values from four independent experiments are shown. C , Ectopic expression of Chk1 reverses HDACi-induced apoptosis but not histone acetylation. A549 cells were transiently transfected with an empty vector (control) or GFP- or FLAG-tagged (GFP-CHK1 or FLAG-CHK1) Chk1 expression plasmid. Forty-eight hours after transfection, cells were cultured without (control, C) or with LBH589 (L) (40 nM) for an additional 24 hour before harvesting for Western blot analysis. Treatment-induced changes in cPARP, acetyl-H4, phospho-CDC2 Y15 , and ectopically expressed GFP-CHK1 or FLAG-CHK1 proteins were determined by Western blot analysis. β-actin expression was used as loading control. Experiments were repeated 3 times, and a representative experiment is shown. The arrows show the position of the GFP-CHK1 and FLAG-CHK1 proteins. D , in primary NSCLC patient samples, Chk1 protein downregulation correlates with increased cPARP ex vivo . Tumor samples were collected with a 23-gauge needle from patient-derived tumors, and cells were treated in duplicate with vehicle (control) or LBH589 (40 nM) for 18 hours. Following treatment, adherent and non-adherent cells were pooled, cell extracts were prepared, and expression levels of cPARP, Chk1, and acetyl-H3 were analyzed by Western blot. β-actin expression was used as loading control. SCC: squamous cell carcinoma; AC: adenocarcinoma.
    Figure Legend Snippet: A549 cells were untreated or treated with LBH589 (40 nM) for various time points. Cell lysates were prepared, and protein expression levels of cPARP, CHK1, Tyr15 phosphorylation of pCDC2 (pCDC2 Y15 ), Ser216 phosphorylation of CDC25C (pCDC25 S216 ), acetyl-H4, and cyclin B1 were determined. B , Quantitative Chk1 mRNA expression analysis. Total RNA was prepared from A549 cells after 24 hours of treatment with 40 nM LBH589 or vehicle. mRNA expression levels were quantified using Real-time PCR analysis. All results were normalized to GAPDH mRNA levels, and the mean and standard deviations values from four independent experiments are shown. C , Ectopic expression of Chk1 reverses HDACi-induced apoptosis but not histone acetylation. A549 cells were transiently transfected with an empty vector (control) or GFP- or FLAG-tagged (GFP-CHK1 or FLAG-CHK1) Chk1 expression plasmid. Forty-eight hours after transfection, cells were cultured without (control, C) or with LBH589 (L) (40 nM) for an additional 24 hour before harvesting for Western blot analysis. Treatment-induced changes in cPARP, acetyl-H4, phospho-CDC2 Y15 , and ectopically expressed GFP-CHK1 or FLAG-CHK1 proteins were determined by Western blot analysis. β-actin expression was used as loading control. Experiments were repeated 3 times, and a representative experiment is shown. The arrows show the position of the GFP-CHK1 and FLAG-CHK1 proteins. D , in primary NSCLC patient samples, Chk1 protein downregulation correlates with increased cPARP ex vivo . Tumor samples were collected with a 23-gauge needle from patient-derived tumors, and cells were treated in duplicate with vehicle (control) or LBH589 (40 nM) for 18 hours. Following treatment, adherent and non-adherent cells were pooled, cell extracts were prepared, and expression levels of cPARP, Chk1, and acetyl-H3 were analyzed by Western blot. β-actin expression was used as loading control. SCC: squamous cell carcinoma; AC: adenocarcinoma.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Ex Vivo, Derivative Assay

    rabbit polyclonal total cdc2  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc rabbit polyclonal total cdc2
    Protein lysates prepared from the ovarian cancer cell lines maintained in growth medium consisting of increasing concentrations of serum and 21% or 3% O 2 were analyzed by Western blot. (A) Compared to O 2 sensitive cell lines, decreased expression of the core components involved in G2/M cell cycle progression <t>CDC2/cyclin</t> B1 complex and its activator CDC25c is observed in the O 2 insensitive cell lines (indicated by asterisk and italics), while the expression of 14-3-3 σ, a protein that inhibits CDC2 is elevated in the O 2 insensitive cell lines. (B) Phosphorylation of RB and Wee1 were monitored as an indicator for CDC2 function because both RB and Wee1 are known targets for phosphorylation by CDC2. Equal loading of protein extracts were monitored by probing the stripped Western blots with the primary antibody for β-actin.
    Rabbit Polyclonal Total Cdc2, 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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    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis"

    Article Title: 14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015864

    Protein lysates prepared from the ovarian cancer cell lines maintained in growth medium consisting of increasing concentrations of serum and 21% or 3% O 2 were analyzed by Western blot. (A) Compared to O 2 sensitive cell lines, decreased expression of the core components involved in G2/M cell cycle progression CDC2/cyclin B1 complex and its activator CDC25c is observed in the O 2 insensitive cell lines (indicated by asterisk and italics), while the expression of 14-3-3 σ, a protein that inhibits CDC2 is elevated in the O 2 insensitive cell lines. (B) Phosphorylation of RB and Wee1 were monitored as an indicator for CDC2 function because both RB and Wee1 are known targets for phosphorylation by CDC2. Equal loading of protein extracts were monitored by probing the stripped Western blots with the primary antibody for β-actin.
    Figure Legend Snippet: Protein lysates prepared from the ovarian cancer cell lines maintained in growth medium consisting of increasing concentrations of serum and 21% or 3% O 2 were analyzed by Western blot. (A) Compared to O 2 sensitive cell lines, decreased expression of the core components involved in G2/M cell cycle progression CDC2/cyclin B1 complex and its activator CDC25c is observed in the O 2 insensitive cell lines (indicated by asterisk and italics), while the expression of 14-3-3 σ, a protein that inhibits CDC2 is elevated in the O 2 insensitive cell lines. (B) Phosphorylation of RB and Wee1 were monitored as an indicator for CDC2 function because both RB and Wee1 are known targets for phosphorylation by CDC2. Equal loading of protein extracts were monitored by probing the stripped Western blots with the primary antibody for β-actin.

    Techniques Used: Western Blot, Expressing

    (A) Cellular localization by immunofluoresence shows that 14-3-3 σ (Green) is located in the cytoplasm and CDC2 (Red) is present in the nucleus (Blue). Compared to O 2 sensitive A2780 cells, the level of 14-3-3 σ is higher and CDC2 is low in the O 2 insensitive HeyA8 cells. In the O 2 sensitive A2780, 14-3-3 σ is localized both in the nucleus and cytoplasm at 21% O 2 . (A dotted yellow line, outlines a representative nuclei to indicate relative localization of 14-3-3 σ and CDC2 in these cells). (B) Western blot analysis of nuclear and cytoplasmic fractions show low levels of 14-3-3 σ in the nucleus compared to cytoplasm, with increased amounts of 14-3-3 σ being present in the cytoplasm of the O 2 insensitive HeyA8 cells. The level of CDC2 is higher both in the nucleus and cytoplasm of the O 2 sensitive A2780, but present in lower amount only in the nucleus of O 2 insensitive HeyA8 cells. Histone H1 and β−actin were used as loading controls for nuclear and cytoplasmic fractions, respectively. (C) Mitotic cells were determined by counting the cells that stained positively for a mitosis specific marker, Phospho-Histone H3 from the total cell population. Mitotic fractions present at 3% or 21% O 2 were counted in both A2780 and HeyA8 and represented as bar graph. A significant increase in mitotic index (p>0.001, indicated by asterisk) was observed in the O 2 sensitive A2780 at 3% O 2 , but not in the O 2 insensitive HeyA8 cells. (D) Over-expression of 14-3-3 σ in the O 2 sensitive A2780 (Western Blot) results in loss of O 2 sensitivity (Bar graph). For the cells transfected with empty vector (mock transfection) or 14-3-3 σ over-expression construct, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2 (ambient), and (E) in the converse experiment performed with O 2 insensitive HeyA8, reducing the levels of 14-3-3 σ by siRNA (Western blot) results in restoration of O 2 sensitivity (Bar graph). For the cells transfected with scrambled siRNA (mock transfection) or siRNA against 14-3-3 σ, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2.
    Figure Legend Snippet: (A) Cellular localization by immunofluoresence shows that 14-3-3 σ (Green) is located in the cytoplasm and CDC2 (Red) is present in the nucleus (Blue). Compared to O 2 sensitive A2780 cells, the level of 14-3-3 σ is higher and CDC2 is low in the O 2 insensitive HeyA8 cells. In the O 2 sensitive A2780, 14-3-3 σ is localized both in the nucleus and cytoplasm at 21% O 2 . (A dotted yellow line, outlines a representative nuclei to indicate relative localization of 14-3-3 σ and CDC2 in these cells). (B) Western blot analysis of nuclear and cytoplasmic fractions show low levels of 14-3-3 σ in the nucleus compared to cytoplasm, with increased amounts of 14-3-3 σ being present in the cytoplasm of the O 2 insensitive HeyA8 cells. The level of CDC2 is higher both in the nucleus and cytoplasm of the O 2 sensitive A2780, but present in lower amount only in the nucleus of O 2 insensitive HeyA8 cells. Histone H1 and β−actin were used as loading controls for nuclear and cytoplasmic fractions, respectively. (C) Mitotic cells were determined by counting the cells that stained positively for a mitosis specific marker, Phospho-Histone H3 from the total cell population. Mitotic fractions present at 3% or 21% O 2 were counted in both A2780 and HeyA8 and represented as bar graph. A significant increase in mitotic index (p>0.001, indicated by asterisk) was observed in the O 2 sensitive A2780 at 3% O 2 , but not in the O 2 insensitive HeyA8 cells. (D) Over-expression of 14-3-3 σ in the O 2 sensitive A2780 (Western Blot) results in loss of O 2 sensitivity (Bar graph). For the cells transfected with empty vector (mock transfection) or 14-3-3 σ over-expression construct, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2 (ambient), and (E) in the converse experiment performed with O 2 insensitive HeyA8, reducing the levels of 14-3-3 σ by siRNA (Western blot) results in restoration of O 2 sensitivity (Bar graph). For the cells transfected with scrambled siRNA (mock transfection) or siRNA against 14-3-3 σ, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2.

    Techniques Used: Western Blot, Staining, Marker, Over Expression, Transfection, Plasmid Preparation, Construct

    (A) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 57 ovarian cancer cell lines. (B) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 205 ovarian tumors. The color codes for overall survival represents overall survival >24 months (blue) and overall survival <24 months (pink). The color codes for tumor stage represent stage I (red), stage II (green), stage III (light-blue) and stage IV (dark-blue). (C). Kaplan-Meier survival curve for the RPPA results comparing the group of ovarian tumors with high Phospho-RB and high 14-3-3 σ or CDC2 (blue line) with other expression profiles (red line).
    Figure Legend Snippet: (A) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 57 ovarian cancer cell lines. (B) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 205 ovarian tumors. The color codes for overall survival represents overall survival >24 months (blue) and overall survival <24 months (pink). The color codes for tumor stage represent stage I (red), stage II (green), stage III (light-blue) and stage IV (dark-blue). (C). Kaplan-Meier survival curve for the RPPA results comparing the group of ovarian tumors with high Phospho-RB and high 14-3-3 σ or CDC2 (blue line) with other expression profiles (red line).

    Techniques Used: Expressing

    total cdk1  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc total cdk1
    ( A ) Approach to identifying candidate kinases that regulate CIC-DUX4 sarcoma survival. Watson et al., 2019: ref. ; Oyama et al., 2017: ref. . ( B ) Reactome pathway analysis identifies CIC-DUX4–dependent cell cycle and DNA replication pathways. ( C ) GSEA reveals cell cycle, DNA replication, and chromosome segregation at CIC-DUX4 targets. ( D ) PamGene array identifies functional kinases that regulate cell cycle and DNA replication in CIC-DUX4 cells. ( E ) Model of WEE1 inhibitory kinase motifs in <t>CDK1</t> and CDK2. ( F ) Schematic of WEE1-regulated cell cycle control.
    Total Cdk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "WEE1 kinase is a therapeutic vulnerability in CIC-DUX4 undifferentiated sarcoma"

    Article Title: WEE1 kinase is a therapeutic vulnerability in CIC-DUX4 undifferentiated sarcoma

    Journal: JCI Insight

    doi: 10.1172/jci.insight.152293

    ( A ) Approach to identifying candidate kinases that regulate CIC-DUX4 sarcoma survival. Watson et al., 2019: ref. ; Oyama et al., 2017: ref. . ( B ) Reactome pathway analysis identifies CIC-DUX4–dependent cell cycle and DNA replication pathways. ( C ) GSEA reveals cell cycle, DNA replication, and chromosome segregation at CIC-DUX4 targets. ( D ) PamGene array identifies functional kinases that regulate cell cycle and DNA replication in CIC-DUX4 cells. ( E ) Model of WEE1 inhibitory kinase motifs in CDK1 and CDK2. ( F ) Schematic of WEE1-regulated cell cycle control.
    Figure Legend Snippet: ( A ) Approach to identifying candidate kinases that regulate CIC-DUX4 sarcoma survival. Watson et al., 2019: ref. ; Oyama et al., 2017: ref. . ( B ) Reactome pathway analysis identifies CIC-DUX4–dependent cell cycle and DNA replication pathways. ( C ) GSEA reveals cell cycle, DNA replication, and chromosome segregation at CIC-DUX4 targets. ( D ) PamGene array identifies functional kinases that regulate cell cycle and DNA replication in CIC-DUX4 cells. ( E ) Model of WEE1 inhibitory kinase motifs in CDK1 and CDK2. ( F ) Schematic of WEE1-regulated cell cycle control.

    Techniques Used: Functional Assay

    ( A ) CTG viability assay of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells treated with adavosertib. Error bars represent SEM; performed in duplicate. Crystal violet assay of NCC_CDS1_X1_C1 ( B ) and NCC_CDS2_C1 ( C ) cells comparing adavosertib (IC 50 dose) and DMSO control. ( D ) Immunoblot analysis of NCC_CDS1_X1_C1 cells treated with adavosertib or DMSO control. Representative figure; performed in duplicate. Lysates were run on parallel gels. FL, full-length; Cl, cleaved. ( E ) Immunoblot analysis of NCC_CDS2_C1 cells treated with adavosertib or DMSO control. Representative figure; performed in duplicate. ( F and G ) Relative caspase-3/7 activity in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells treated with adavosertib versus control. One-way ANOVA; performed in triplicate. ( H and I ) Immunoblot analysis of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing 2 independent WEE1 siRNAs. Representative figure; performed in duplicate. ( J and K ) CTG viability assay comparing 2 independent WEE1 siRNAs with scramble control in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. One-way ANOVA; performed in triplicate. ( L and M ) Viability assay comparing 2 independent WEE1 siRNAs with scramble control (siScrm) in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. One-way ANOVA; performed in duplicate. ( N and O ) Adavosertib IC 50 dose in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing siCDK1, siCDK2, or siCDK1 and siCDK2. One-way ANOVA; performed in triplicate. ( P ) Relative viability comparing NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing CDK1 WT or CDK1 AF versus vector control. One-way ANOVA. Error bars represent SEM; performed in triplicate. ( Q ) Adavosertib IC 50 dose in NCC_CDS2_C1 cells expressing siCCNE1 or siCtrl. Performed in triplicate. * P <0.001, Student’s t test. ( R ) Adavosertib IC 50 dose in NCC_CDS1_X1_C1 cells expressing si CCNE1 , si CCNE2 , si CCNE1 , and si CCNE2 compared with siCtrl; performed in triplicate. * P <0.001, 1-way ANOVA.
    Figure Legend Snippet: ( A ) CTG viability assay of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells treated with adavosertib. Error bars represent SEM; performed in duplicate. Crystal violet assay of NCC_CDS1_X1_C1 ( B ) and NCC_CDS2_C1 ( C ) cells comparing adavosertib (IC 50 dose) and DMSO control. ( D ) Immunoblot analysis of NCC_CDS1_X1_C1 cells treated with adavosertib or DMSO control. Representative figure; performed in duplicate. Lysates were run on parallel gels. FL, full-length; Cl, cleaved. ( E ) Immunoblot analysis of NCC_CDS2_C1 cells treated with adavosertib or DMSO control. Representative figure; performed in duplicate. ( F and G ) Relative caspase-3/7 activity in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells treated with adavosertib versus control. One-way ANOVA; performed in triplicate. ( H and I ) Immunoblot analysis of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing 2 independent WEE1 siRNAs. Representative figure; performed in duplicate. ( J and K ) CTG viability assay comparing 2 independent WEE1 siRNAs with scramble control in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. One-way ANOVA; performed in triplicate. ( L and M ) Viability assay comparing 2 independent WEE1 siRNAs with scramble control (siScrm) in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. One-way ANOVA; performed in duplicate. ( N and O ) Adavosertib IC 50 dose in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing siCDK1, siCDK2, or siCDK1 and siCDK2. One-way ANOVA; performed in triplicate. ( P ) Relative viability comparing NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing CDK1 WT or CDK1 AF versus vector control. One-way ANOVA. Error bars represent SEM; performed in triplicate. ( Q ) Adavosertib IC 50 dose in NCC_CDS2_C1 cells expressing siCCNE1 or siCtrl. Performed in triplicate. * P <0.001, Student’s t test. ( R ) Adavosertib IC 50 dose in NCC_CDS1_X1_C1 cells expressing si CCNE1 , si CCNE2 , si CCNE1 , and si CCNE2 compared with siCtrl; performed in triplicate. * P <0.001, 1-way ANOVA.

    Techniques Used: Viability Assay, Crystal Violet Assay, Western Blot, Activity Assay, Expressing, Plasmid Preparation

    total cdc2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc total cdc2
    (A) Immunoblot analysis for the G2-M cell cycle markers: pCDC2(T15), <t>total-CDC2,</t> and cyclin B1 on the Icmt +/+ (WT) and Icmt −/− (N1, N2, and N3) isogenic cell lines harvested after growing under standard culture conditions before reach confluency and processed for SDS–PAGE. The numbers below each marker are the densitometry quantification of band intensity. (B) Cell cycle distribution of the isogenic cells in the baseline state without synchronization (top row), and following double thymidine synchronization (0 h, middle row) and 8 h post-release (bottom row). The red vertical lines mark the G1 and G2/M peaks. The cells were prepared by standard propidium iodide staining method for cell cycle analysis, as described in the Materials and Methods section. (C) Flow cytometry analysis of Citrine-Geminin and mCherry-Cdt1 in the isogenic cell lines stably expressing these cell cycle markers. Analysis was performed at the baseline state without synchronization (top row), at the time of release from the double thymidine block (middle row, 0 h), and 8 h after release from double thymidine block (bottom row). (B) These cells were subjected to the same preparation conditions as in (B) before standard flow cytometry analysis. The experiments were performed three times with similar findings.
    Total Cdc2, 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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    1) Product Images from "Suppression of isoprenylcysteine carboxylmethyltransferase compromises DNA damage repair"

    Article Title: Suppression of isoprenylcysteine carboxylmethyltransferase compromises DNA damage repair

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202101144

    (A) Immunoblot analysis for the G2-M cell cycle markers: pCDC2(T15), total-CDC2, and cyclin B1 on the Icmt +/+ (WT) and Icmt −/− (N1, N2, and N3) isogenic cell lines harvested after growing under standard culture conditions before reach confluency and processed for SDS–PAGE. The numbers below each marker are the densitometry quantification of band intensity. (B) Cell cycle distribution of the isogenic cells in the baseline state without synchronization (top row), and following double thymidine synchronization (0 h, middle row) and 8 h post-release (bottom row). The red vertical lines mark the G1 and G2/M peaks. The cells were prepared by standard propidium iodide staining method for cell cycle analysis, as described in the Materials and Methods section. (C) Flow cytometry analysis of Citrine-Geminin and mCherry-Cdt1 in the isogenic cell lines stably expressing these cell cycle markers. Analysis was performed at the baseline state without synchronization (top row), at the time of release from the double thymidine block (middle row, 0 h), and 8 h after release from double thymidine block (bottom row). (B) These cells were subjected to the same preparation conditions as in (B) before standard flow cytometry analysis. The experiments were performed three times with similar findings.
    Figure Legend Snippet: (A) Immunoblot analysis for the G2-M cell cycle markers: pCDC2(T15), total-CDC2, and cyclin B1 on the Icmt +/+ (WT) and Icmt −/− (N1, N2, and N3) isogenic cell lines harvested after growing under standard culture conditions before reach confluency and processed for SDS–PAGE. The numbers below each marker are the densitometry quantification of band intensity. (B) Cell cycle distribution of the isogenic cells in the baseline state without synchronization (top row), and following double thymidine synchronization (0 h, middle row) and 8 h post-release (bottom row). The red vertical lines mark the G1 and G2/M peaks. The cells were prepared by standard propidium iodide staining method for cell cycle analysis, as described in the Materials and Methods section. (C) Flow cytometry analysis of Citrine-Geminin and mCherry-Cdt1 in the isogenic cell lines stably expressing these cell cycle markers. Analysis was performed at the baseline state without synchronization (top row), at the time of release from the double thymidine block (middle row, 0 h), and 8 h after release from double thymidine block (bottom row). (B) These cells were subjected to the same preparation conditions as in (B) before standard flow cytometry analysis. The experiments were performed three times with similar findings.

    Techniques Used: Western Blot, SDS Page, Marker, Staining, Cell Cycle Assay, Flow Cytometry, Stable Transfection, Expressing, Blocking Assay

    total cdk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc total cdk1
    (A) Approach to identify candidate kinases that regulate CIC-DUX4 survival. (B) Reactome pathway analysis identifies CIC-DUX4-dependent cell cycle and DNA replication pathways. (C) Gene Set Enrichment Analysis (GSEA) reveals cell cycle, DNA replication, and chromosome segregation at CIC-DUX4 targets. (D) PamGene array identifies functional kinases that regulate cell cycle and DNA replication in CIC-DUX4 cells. (E) Model of Wee1 inhibitory kinase motifs in <t>CDK1</t> and CDK2. (F) Schematic of WEE1-regulated cell cycle control.
    Total Cdk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "WEE1 kinase is a therapeutic vulnerability in CIC-DUX4 undifferentiated sarcoma"

    Article Title: WEE1 kinase is a therapeutic vulnerability in CIC-DUX4 undifferentiated sarcoma

    Journal: bioRxiv

    doi: 10.1101/2021.06.21.448439

    (A) Approach to identify candidate kinases that regulate CIC-DUX4 survival. (B) Reactome pathway analysis identifies CIC-DUX4-dependent cell cycle and DNA replication pathways. (C) Gene Set Enrichment Analysis (GSEA) reveals cell cycle, DNA replication, and chromosome segregation at CIC-DUX4 targets. (D) PamGene array identifies functional kinases that regulate cell cycle and DNA replication in CIC-DUX4 cells. (E) Model of Wee1 inhibitory kinase motifs in CDK1 and CDK2. (F) Schematic of WEE1-regulated cell cycle control.
    Figure Legend Snippet: (A) Approach to identify candidate kinases that regulate CIC-DUX4 survival. (B) Reactome pathway analysis identifies CIC-DUX4-dependent cell cycle and DNA replication pathways. (C) Gene Set Enrichment Analysis (GSEA) reveals cell cycle, DNA replication, and chromosome segregation at CIC-DUX4 targets. (D) PamGene array identifies functional kinases that regulate cell cycle and DNA replication in CIC-DUX4 cells. (E) Model of Wee1 inhibitory kinase motifs in CDK1 and CDK2. (F) Schematic of WEE1-regulated cell cycle control.

    Techniques Used: Functional Assay

    (A) Cell-Titer Glo viability assay of NCC_CDS1_X1_C1 and NCC_CDS2_C1 treated with adavosertib. Error Bars represent SEM. Performed in duplicate. Crystal Violet assay of NCC_CDS1_X1_C1 (B) and NCC_CDS2_C1 (C) cells comparing adavosertib (IC 50 dose) and DMSO control. (D-E) Immunoblot analysis of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells treated with adavosertib or DMSO control. Representative figure; performed in duplicate. (F-G) Relative caspase 3/7 activity in NCC_CDS1_X1_C1 and NCC_CDS2_C1 treated with adavosertib vs control. P-value, 1-way ANOVA. Performed in triplicate (H-I) Immunoblot analysis of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing two independent Wee1 siRNAs. Representative figure; performed in duplicate. (J-K) Cell-Titer Glo viability assay comparing two independent Wee1 siRNAs to scramble control in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. P-value, 1-way ANOVA. Performed in triplicate. (L-M) Viability assay comparing two independent Wee1 siRNAs to scramble control in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. P-value, 1-way ANOVA. Performed in duplicate. (N-O) Adavosertib IC 50 dose in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing siCDK1, siCDK2, or siCDK1 and siCDK2. P-value, 1-way ANOVA. Performed in triplicate. (P) Relative viability comparing NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing CDK1 WT or CDK1 AF versus vector control. P-value, 1-way ANOVA. Error bars represent SEM. Performed in triplicate.
    Figure Legend Snippet: (A) Cell-Titer Glo viability assay of NCC_CDS1_X1_C1 and NCC_CDS2_C1 treated with adavosertib. Error Bars represent SEM. Performed in duplicate. Crystal Violet assay of NCC_CDS1_X1_C1 (B) and NCC_CDS2_C1 (C) cells comparing adavosertib (IC 50 dose) and DMSO control. (D-E) Immunoblot analysis of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells treated with adavosertib or DMSO control. Representative figure; performed in duplicate. (F-G) Relative caspase 3/7 activity in NCC_CDS1_X1_C1 and NCC_CDS2_C1 treated with adavosertib vs control. P-value, 1-way ANOVA. Performed in triplicate (H-I) Immunoblot analysis of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing two independent Wee1 siRNAs. Representative figure; performed in duplicate. (J-K) Cell-Titer Glo viability assay comparing two independent Wee1 siRNAs to scramble control in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. P-value, 1-way ANOVA. Performed in triplicate. (L-M) Viability assay comparing two independent Wee1 siRNAs to scramble control in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. P-value, 1-way ANOVA. Performed in duplicate. (N-O) Adavosertib IC 50 dose in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing siCDK1, siCDK2, or siCDK1 and siCDK2. P-value, 1-way ANOVA. Performed in triplicate. (P) Relative viability comparing NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing CDK1 WT or CDK1 AF versus vector control. P-value, 1-way ANOVA. Error bars represent SEM. Performed in triplicate.

    Techniques Used: Viability Assay, Crystal Violet Assay, Western Blot, Activity Assay, Expressing, Plasmid Preparation

    anti mouse total cdk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti mouse total cdk1
    Anti Mouse Total Cdk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti mouse total cdk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti mouse total cdk1
    Anti Mouse Total Cdk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    total cdc2 antibodies  (Cell Signaling Technology Inc)


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    total cdk1  (Cell Signaling Technology Inc)


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    The gene list for the differentially expressed genes of cervical cancer (CxCa-DEGs) and the cervical cancer signature (CxCa-Sig)
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    1) Product Images from "Bioinformatics and in vitro experimental analyses identify the selective therapeutic potential of interferon gamma and apigenin against cervical squamous cell carcinoma and adenocarcinoma"

    Article Title: Bioinformatics and in vitro experimental analyses identify the selective therapeutic potential of interferon gamma and apigenin against cervical squamous cell carcinoma and adenocarcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17574

    The gene list for the differentially expressed genes of cervical cancer (CxCa-DEGs) and the cervical cancer signature (CxCa-Sig)
    Figure Legend Snippet: The gene list for the differentially expressed genes of cervical cancer (CxCa-DEGs) and the cervical cancer signature (CxCa-Sig)

    Techniques Used:

    total cdc2 antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc total cdc2 antibodies
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    A , A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of <t>CDC2</t> (pCDC2 Y15 ), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. B , PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 Y15 ), serine-216 phosphorylation of CDC25C (pCDC25c S216 ), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. C , drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. D , PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. E , A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 Y15 , and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.
    Rabbit Total Cdc2, 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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    Protein lysates prepared from the ovarian cancer cell lines maintained in growth medium consisting of increasing concentrations of serum and 21% or 3% O 2 were analyzed by Western blot. (A) Compared to O 2 sensitive cell lines, decreased expression of the core components involved in G2/M cell cycle progression <t>CDC2/cyclin</t> B1 complex and its activator CDC25c is observed in the O 2 insensitive cell lines (indicated by asterisk and italics), while the expression of 14-3-3 σ, a protein that inhibits CDC2 is elevated in the O 2 insensitive cell lines. (B) Phosphorylation of RB and Wee1 were monitored as an indicator for CDC2 function because both RB and Wee1 are known targets for phosphorylation by CDC2. Equal loading of protein extracts were monitored by probing the stripped Western blots with the primary antibody for β-actin.
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    ( A ) Approach to identifying candidate kinases that regulate CIC-DUX4 sarcoma survival. Watson et al., 2019: ref. ; Oyama et al., 2017: ref. . ( B ) Reactome pathway analysis identifies CIC-DUX4–dependent cell cycle and DNA replication pathways. ( C ) GSEA reveals cell cycle, DNA replication, and chromosome segregation at CIC-DUX4 targets. ( D ) PamGene array identifies functional kinases that regulate cell cycle and DNA replication in CIC-DUX4 cells. ( E ) Model of WEE1 inhibitory kinase motifs in <t>CDK1</t> and CDK2. ( F ) Schematic of WEE1-regulated cell cycle control.
    Total Cdk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Immunoblot analysis for the G2-M cell cycle markers: pCDC2(T15), <t>total-CDC2,</t> and cyclin B1 on the Icmt +/+ (WT) and Icmt −/− (N1, N2, and N3) isogenic cell lines harvested after growing under standard culture conditions before reach confluency and processed for SDS–PAGE. The numbers below each marker are the densitometry quantification of band intensity. (B) Cell cycle distribution of the isogenic cells in the baseline state without synchronization (top row), and following double thymidine synchronization (0 h, middle row) and 8 h post-release (bottom row). The red vertical lines mark the G1 and G2/M peaks. The cells were prepared by standard propidium iodide staining method for cell cycle analysis, as described in the Materials and Methods section. (C) Flow cytometry analysis of Citrine-Geminin and mCherry-Cdt1 in the isogenic cell lines stably expressing these cell cycle markers. Analysis was performed at the baseline state without synchronization (top row), at the time of release from the double thymidine block (middle row, 0 h), and 8 h after release from double thymidine block (bottom row). (B) These cells were subjected to the same preparation conditions as in (B) before standard flow cytometry analysis. The experiments were performed three times with similar findings.
    Total Cdc2, 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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    99
    Cell Signaling Technology Inc anti mouse total cdk1
    (A) Immunoblot analysis for the G2-M cell cycle markers: pCDC2(T15), <t>total-CDC2,</t> and cyclin B1 on the Icmt +/+ (WT) and Icmt −/− (N1, N2, and N3) isogenic cell lines harvested after growing under standard culture conditions before reach confluency and processed for SDS–PAGE. The numbers below each marker are the densitometry quantification of band intensity. (B) Cell cycle distribution of the isogenic cells in the baseline state without synchronization (top row), and following double thymidine synchronization (0 h, middle row) and 8 h post-release (bottom row). The red vertical lines mark the G1 and G2/M peaks. The cells were prepared by standard propidium iodide staining method for cell cycle analysis, as described in the Materials and Methods section. (C) Flow cytometry analysis of Citrine-Geminin and mCherry-Cdt1 in the isogenic cell lines stably expressing these cell cycle markers. Analysis was performed at the baseline state without synchronization (top row), at the time of release from the double thymidine block (middle row, 0 h), and 8 h after release from double thymidine block (bottom row). (B) These cells were subjected to the same preparation conditions as in (B) before standard flow cytometry analysis. The experiments were performed three times with similar findings.
    Anti Mouse Total Cdk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    total cdc2 antibodies  (Cell Signaling Technology Inc)
    96
    Cell Signaling Technology Inc total cdc2 antibodies
    (A) Immunoblot analysis for the G2-M cell cycle markers: pCDC2(T15), <t>total-CDC2,</t> and cyclin B1 on the Icmt +/+ (WT) and Icmt −/− (N1, N2, and N3) isogenic cell lines harvested after growing under standard culture conditions before reach confluency and processed for SDS–PAGE. The numbers below each marker are the densitometry quantification of band intensity. (B) Cell cycle distribution of the isogenic cells in the baseline state without synchronization (top row), and following double thymidine synchronization (0 h, middle row) and 8 h post-release (bottom row). The red vertical lines mark the G1 and G2/M peaks. The cells were prepared by standard propidium iodide staining method for cell cycle analysis, as described in the Materials and Methods section. (C) Flow cytometry analysis of Citrine-Geminin and mCherry-Cdt1 in the isogenic cell lines stably expressing these cell cycle markers. Analysis was performed at the baseline state without synchronization (top row), at the time of release from the double thymidine block (middle row, 0 h), and 8 h after release from double thymidine block (bottom row). (B) These cells were subjected to the same preparation conditions as in (B) before standard flow cytometry analysis. The experiments were performed three times with similar findings.
    Total Cdc2 Antibodies, 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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    Image Search Results


    A , A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of CDC2 (pCDC2 Y15 ), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. B , PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 Y15 ), serine-216 phosphorylation of CDC25C (pCDC25c S216 ), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. C , drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. D , PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. E , A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 Y15 , and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.

    Journal: PLoS ONE

    Article Title: Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.1371/journal.pone.0014335

    Figure Lengend Snippet: A , A549, PC9, H1299, H292, H358, H441 and HCC827 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM for 24 hours and expression levels of cPARP, phosphorylation of CDC2 (pCDC2 Y15 ), CHK1, and acetylated histone H4 (acetyl-H4) were determined by Western blot and quantitated using AlphaEase software. β-actin was used as loading control. B , PC9 and A549 cells were cultured in the presence of vehicle (C), or LBH589 (LBH) 40 nM, or scriptaid (S) 1 µM for 24 hours and expression levels of cPARP, tyrosine-15 phosphorylation of CDC2 (pCDC2 Y15 ), serine-216 phosphorylation of CDC25C (pCDC25c S216 ), CDC25A (T-CDC25A), CDC25C (T-CDC25C) and CDC2 (T-CDC2), acetylated histone H4 (acetyl-H4), and cyclin B1 were determined by Western blot analysis. β-actin was used as loading control. C , drug-mediated changes in the expression of CHK1, CHK2, AKT, and c-RAF were determined by Western blot analysis. β-actin was used as loading control. D , PC9 or A549 cells were treated with or without 40 nM LBH589 and analyzed for annexin positive cells using the BD Annexin V-FITC/7-AAD Flow Cytometry kit. E , A549 cells were treated with MS-275 (MS), (500 nM), valproic acid (VA) (1 Mm), or apicidin (Api) (400 nM) for 24 h. Expression levels of cPARP, CHK1, pCDC2 Y15 , and β-actin were determined by Western blot analysis. All experiments were repeated at least three times.

    Article Snippet: Mouse total Chk1, mouse total Chk2, rabbit cdc2 Y15P, rabbit cdc25c S216P, rabbit acetylated histones H3 and H4 (acetyl-H3 or acetyl-H4), rabbit total cdc2, rabbit total cdc25c, rabbit total cdc25a, rabbit total histone H3, rabbit cPARP, rabbit total AKT, rabbit H3 S10P, and rabbit total c-RAF antibodies were obtained from Cell Signaling Technology (Watertown, MA).

    Techniques: Cell Culture, Expressing, Western Blot, Software, Flow Cytometry

    A549 cells were untreated or treated with LBH589 (40 nM) for various time points. Cell lysates were prepared, and protein expression levels of cPARP, CHK1, Tyr15 phosphorylation of pCDC2 (pCDC2 Y15 ), Ser216 phosphorylation of CDC25C (pCDC25 S216 ), acetyl-H4, and cyclin B1 were determined. B , Quantitative Chk1 mRNA expression analysis. Total RNA was prepared from A549 cells after 24 hours of treatment with 40 nM LBH589 or vehicle. mRNA expression levels were quantified using Real-time PCR analysis. All results were normalized to GAPDH mRNA levels, and the mean and standard deviations values from four independent experiments are shown. C , Ectopic expression of Chk1 reverses HDACi-induced apoptosis but not histone acetylation. A549 cells were transiently transfected with an empty vector (control) or GFP- or FLAG-tagged (GFP-CHK1 or FLAG-CHK1) Chk1 expression plasmid. Forty-eight hours after transfection, cells were cultured without (control, C) or with LBH589 (L) (40 nM) for an additional 24 hour before harvesting for Western blot analysis. Treatment-induced changes in cPARP, acetyl-H4, phospho-CDC2 Y15 , and ectopically expressed GFP-CHK1 or FLAG-CHK1 proteins were determined by Western blot analysis. β-actin expression was used as loading control. Experiments were repeated 3 times, and a representative experiment is shown. The arrows show the position of the GFP-CHK1 and FLAG-CHK1 proteins. D , in primary NSCLC patient samples, Chk1 protein downregulation correlates with increased cPARP ex vivo . Tumor samples were collected with a 23-gauge needle from patient-derived tumors, and cells were treated in duplicate with vehicle (control) or LBH589 (40 nM) for 18 hours. Following treatment, adherent and non-adherent cells were pooled, cell extracts were prepared, and expression levels of cPARP, Chk1, and acetyl-H3 were analyzed by Western blot. β-actin expression was used as loading control. SCC: squamous cell carcinoma; AC: adenocarcinoma.

    Journal: PLoS ONE

    Article Title: Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells

    doi: 10.1371/journal.pone.0014335

    Figure Lengend Snippet: A549 cells were untreated or treated with LBH589 (40 nM) for various time points. Cell lysates were prepared, and protein expression levels of cPARP, CHK1, Tyr15 phosphorylation of pCDC2 (pCDC2 Y15 ), Ser216 phosphorylation of CDC25C (pCDC25 S216 ), acetyl-H4, and cyclin B1 were determined. B , Quantitative Chk1 mRNA expression analysis. Total RNA was prepared from A549 cells after 24 hours of treatment with 40 nM LBH589 or vehicle. mRNA expression levels were quantified using Real-time PCR analysis. All results were normalized to GAPDH mRNA levels, and the mean and standard deviations values from four independent experiments are shown. C , Ectopic expression of Chk1 reverses HDACi-induced apoptosis but not histone acetylation. A549 cells were transiently transfected with an empty vector (control) or GFP- or FLAG-tagged (GFP-CHK1 or FLAG-CHK1) Chk1 expression plasmid. Forty-eight hours after transfection, cells were cultured without (control, C) or with LBH589 (L) (40 nM) for an additional 24 hour before harvesting for Western blot analysis. Treatment-induced changes in cPARP, acetyl-H4, phospho-CDC2 Y15 , and ectopically expressed GFP-CHK1 or FLAG-CHK1 proteins were determined by Western blot analysis. β-actin expression was used as loading control. Experiments were repeated 3 times, and a representative experiment is shown. The arrows show the position of the GFP-CHK1 and FLAG-CHK1 proteins. D , in primary NSCLC patient samples, Chk1 protein downregulation correlates with increased cPARP ex vivo . Tumor samples were collected with a 23-gauge needle from patient-derived tumors, and cells were treated in duplicate with vehicle (control) or LBH589 (40 nM) for 18 hours. Following treatment, adherent and non-adherent cells were pooled, cell extracts were prepared, and expression levels of cPARP, Chk1, and acetyl-H3 were analyzed by Western blot. β-actin expression was used as loading control. SCC: squamous cell carcinoma; AC: adenocarcinoma.

    Article Snippet: Mouse total Chk1, mouse total Chk2, rabbit cdc2 Y15P, rabbit cdc25c S216P, rabbit acetylated histones H3 and H4 (acetyl-H3 or acetyl-H4), rabbit total cdc2, rabbit total cdc25c, rabbit total cdc25a, rabbit total histone H3, rabbit cPARP, rabbit total AKT, rabbit H3 S10P, and rabbit total c-RAF antibodies were obtained from Cell Signaling Technology (Watertown, MA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Ex Vivo, Derivative Assay

    Protein lysates prepared from the ovarian cancer cell lines maintained in growth medium consisting of increasing concentrations of serum and 21% or 3% O 2 were analyzed by Western blot. (A) Compared to O 2 sensitive cell lines, decreased expression of the core components involved in G2/M cell cycle progression CDC2/cyclin B1 complex and its activator CDC25c is observed in the O 2 insensitive cell lines (indicated by asterisk and italics), while the expression of 14-3-3 σ, a protein that inhibits CDC2 is elevated in the O 2 insensitive cell lines. (B) Phosphorylation of RB and Wee1 were monitored as an indicator for CDC2 function because both RB and Wee1 are known targets for phosphorylation by CDC2. Equal loading of protein extracts were monitored by probing the stripped Western blots with the primary antibody for β-actin.

    Journal: PLoS ONE

    Article Title: 14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

    doi: 10.1371/journal.pone.0015864

    Figure Lengend Snippet: Protein lysates prepared from the ovarian cancer cell lines maintained in growth medium consisting of increasing concentrations of serum and 21% or 3% O 2 were analyzed by Western blot. (A) Compared to O 2 sensitive cell lines, decreased expression of the core components involved in G2/M cell cycle progression CDC2/cyclin B1 complex and its activator CDC25c is observed in the O 2 insensitive cell lines (indicated by asterisk and italics), while the expression of 14-3-3 σ, a protein that inhibits CDC2 is elevated in the O 2 insensitive cell lines. (B) Phosphorylation of RB and Wee1 were monitored as an indicator for CDC2 function because both RB and Wee1 are known targets for phosphorylation by CDC2. Equal loading of protein extracts were monitored by probing the stripped Western blots with the primary antibody for β-actin.

    Article Snippet: The primary antibodies used were mouse monoclonal 14-3-3 σ at 1.0 µg/mL (Upstate) and rabbit polyclonal total CDC2 at 1∶1000 (Cell Signaling).

    Techniques: Western Blot, Expressing

    (A) Cellular localization by immunofluoresence shows that 14-3-3 σ (Green) is located in the cytoplasm and CDC2 (Red) is present in the nucleus (Blue). Compared to O 2 sensitive A2780 cells, the level of 14-3-3 σ is higher and CDC2 is low in the O 2 insensitive HeyA8 cells. In the O 2 sensitive A2780, 14-3-3 σ is localized both in the nucleus and cytoplasm at 21% O 2 . (A dotted yellow line, outlines a representative nuclei to indicate relative localization of 14-3-3 σ and CDC2 in these cells). (B) Western blot analysis of nuclear and cytoplasmic fractions show low levels of 14-3-3 σ in the nucleus compared to cytoplasm, with increased amounts of 14-3-3 σ being present in the cytoplasm of the O 2 insensitive HeyA8 cells. The level of CDC2 is higher both in the nucleus and cytoplasm of the O 2 sensitive A2780, but present in lower amount only in the nucleus of O 2 insensitive HeyA8 cells. Histone H1 and β−actin were used as loading controls for nuclear and cytoplasmic fractions, respectively. (C) Mitotic cells were determined by counting the cells that stained positively for a mitosis specific marker, Phospho-Histone H3 from the total cell population. Mitotic fractions present at 3% or 21% O 2 were counted in both A2780 and HeyA8 and represented as bar graph. A significant increase in mitotic index (p>0.001, indicated by asterisk) was observed in the O 2 sensitive A2780 at 3% O 2 , but not in the O 2 insensitive HeyA8 cells. (D) Over-expression of 14-3-3 σ in the O 2 sensitive A2780 (Western Blot) results in loss of O 2 sensitivity (Bar graph). For the cells transfected with empty vector (mock transfection) or 14-3-3 σ over-expression construct, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2 (ambient), and (E) in the converse experiment performed with O 2 insensitive HeyA8, reducing the levels of 14-3-3 σ by siRNA (Western blot) results in restoration of O 2 sensitivity (Bar graph). For the cells transfected with scrambled siRNA (mock transfection) or siRNA against 14-3-3 σ, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2.

    Journal: PLoS ONE

    Article Title: 14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

    doi: 10.1371/journal.pone.0015864

    Figure Lengend Snippet: (A) Cellular localization by immunofluoresence shows that 14-3-3 σ (Green) is located in the cytoplasm and CDC2 (Red) is present in the nucleus (Blue). Compared to O 2 sensitive A2780 cells, the level of 14-3-3 σ is higher and CDC2 is low in the O 2 insensitive HeyA8 cells. In the O 2 sensitive A2780, 14-3-3 σ is localized both in the nucleus and cytoplasm at 21% O 2 . (A dotted yellow line, outlines a representative nuclei to indicate relative localization of 14-3-3 σ and CDC2 in these cells). (B) Western blot analysis of nuclear and cytoplasmic fractions show low levels of 14-3-3 σ in the nucleus compared to cytoplasm, with increased amounts of 14-3-3 σ being present in the cytoplasm of the O 2 insensitive HeyA8 cells. The level of CDC2 is higher both in the nucleus and cytoplasm of the O 2 sensitive A2780, but present in lower amount only in the nucleus of O 2 insensitive HeyA8 cells. Histone H1 and β−actin were used as loading controls for nuclear and cytoplasmic fractions, respectively. (C) Mitotic cells were determined by counting the cells that stained positively for a mitosis specific marker, Phospho-Histone H3 from the total cell population. Mitotic fractions present at 3% or 21% O 2 were counted in both A2780 and HeyA8 and represented as bar graph. A significant increase in mitotic index (p>0.001, indicated by asterisk) was observed in the O 2 sensitive A2780 at 3% O 2 , but not in the O 2 insensitive HeyA8 cells. (D) Over-expression of 14-3-3 σ in the O 2 sensitive A2780 (Western Blot) results in loss of O 2 sensitivity (Bar graph). For the cells transfected with empty vector (mock transfection) or 14-3-3 σ over-expression construct, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2 (ambient), and (E) in the converse experiment performed with O 2 insensitive HeyA8, reducing the levels of 14-3-3 σ by siRNA (Western blot) results in restoration of O 2 sensitivity (Bar graph). For the cells transfected with scrambled siRNA (mock transfection) or siRNA against 14-3-3 σ, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2.

    Article Snippet: The primary antibodies used were mouse monoclonal 14-3-3 σ at 1.0 µg/mL (Upstate) and rabbit polyclonal total CDC2 at 1∶1000 (Cell Signaling).

    Techniques: Western Blot, Staining, Marker, Over Expression, Transfection, Plasmid Preparation, Construct

    (A) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 57 ovarian cancer cell lines. (B) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 205 ovarian tumors. The color codes for overall survival represents overall survival >24 months (blue) and overall survival <24 months (pink). The color codes for tumor stage represent stage I (red), stage II (green), stage III (light-blue) and stage IV (dark-blue). (C). Kaplan-Meier survival curve for the RPPA results comparing the group of ovarian tumors with high Phospho-RB and high 14-3-3 σ or CDC2 (blue line) with other expression profiles (red line).

    Journal: PLoS ONE

    Article Title: 14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

    doi: 10.1371/journal.pone.0015864

    Figure Lengend Snippet: (A) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 57 ovarian cancer cell lines. (B) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 205 ovarian tumors. The color codes for overall survival represents overall survival >24 months (blue) and overall survival <24 months (pink). The color codes for tumor stage represent stage I (red), stage II (green), stage III (light-blue) and stage IV (dark-blue). (C). Kaplan-Meier survival curve for the RPPA results comparing the group of ovarian tumors with high Phospho-RB and high 14-3-3 σ or CDC2 (blue line) with other expression profiles (red line).

    Article Snippet: The primary antibodies used were mouse monoclonal 14-3-3 σ at 1.0 µg/mL (Upstate) and rabbit polyclonal total CDC2 at 1∶1000 (Cell Signaling).

    Techniques: Expressing

    ( A ) Approach to identifying candidate kinases that regulate CIC-DUX4 sarcoma survival. Watson et al., 2019: ref. ; Oyama et al., 2017: ref. . ( B ) Reactome pathway analysis identifies CIC-DUX4–dependent cell cycle and DNA replication pathways. ( C ) GSEA reveals cell cycle, DNA replication, and chromosome segregation at CIC-DUX4 targets. ( D ) PamGene array identifies functional kinases that regulate cell cycle and DNA replication in CIC-DUX4 cells. ( E ) Model of WEE1 inhibitory kinase motifs in CDK1 and CDK2. ( F ) Schematic of WEE1-regulated cell cycle control.

    Journal: JCI Insight

    Article Title: WEE1 kinase is a therapeutic vulnerability in CIC-DUX4 undifferentiated sarcoma

    doi: 10.1172/jci.insight.152293

    Figure Lengend Snippet: ( A ) Approach to identifying candidate kinases that regulate CIC-DUX4 sarcoma survival. Watson et al., 2019: ref. ; Oyama et al., 2017: ref. . ( B ) Reactome pathway analysis identifies CIC-DUX4–dependent cell cycle and DNA replication pathways. ( C ) GSEA reveals cell cycle, DNA replication, and chromosome segregation at CIC-DUX4 targets. ( D ) PamGene array identifies functional kinases that regulate cell cycle and DNA replication in CIC-DUX4 cells. ( E ) Model of WEE1 inhibitory kinase motifs in CDK1 and CDK2. ( F ) Schematic of WEE1-regulated cell cycle control.

    Article Snippet: Antibodies to PARP, phosphorylated (Y15) CDK1, HSP90, and total CDK1 were purchased from CST.

    Techniques: Functional Assay

    ( A ) CTG viability assay of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells treated with adavosertib. Error bars represent SEM; performed in duplicate. Crystal violet assay of NCC_CDS1_X1_C1 ( B ) and NCC_CDS2_C1 ( C ) cells comparing adavosertib (IC 50 dose) and DMSO control. ( D ) Immunoblot analysis of NCC_CDS1_X1_C1 cells treated with adavosertib or DMSO control. Representative figure; performed in duplicate. Lysates were run on parallel gels. FL, full-length; Cl, cleaved. ( E ) Immunoblot analysis of NCC_CDS2_C1 cells treated with adavosertib or DMSO control. Representative figure; performed in duplicate. ( F and G ) Relative caspase-3/7 activity in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells treated with adavosertib versus control. One-way ANOVA; performed in triplicate. ( H and I ) Immunoblot analysis of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing 2 independent WEE1 siRNAs. Representative figure; performed in duplicate. ( J and K ) CTG viability assay comparing 2 independent WEE1 siRNAs with scramble control in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. One-way ANOVA; performed in triplicate. ( L and M ) Viability assay comparing 2 independent WEE1 siRNAs with scramble control (siScrm) in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. One-way ANOVA; performed in duplicate. ( N and O ) Adavosertib IC 50 dose in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing siCDK1, siCDK2, or siCDK1 and siCDK2. One-way ANOVA; performed in triplicate. ( P ) Relative viability comparing NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing CDK1 WT or CDK1 AF versus vector control. One-way ANOVA. Error bars represent SEM; performed in triplicate. ( Q ) Adavosertib IC 50 dose in NCC_CDS2_C1 cells expressing siCCNE1 or siCtrl. Performed in triplicate. * P <0.001, Student’s t test. ( R ) Adavosertib IC 50 dose in NCC_CDS1_X1_C1 cells expressing si CCNE1 , si CCNE2 , si CCNE1 , and si CCNE2 compared with siCtrl; performed in triplicate. * P <0.001, 1-way ANOVA.

    Journal: JCI Insight

    Article Title: WEE1 kinase is a therapeutic vulnerability in CIC-DUX4 undifferentiated sarcoma

    doi: 10.1172/jci.insight.152293

    Figure Lengend Snippet: ( A ) CTG viability assay of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells treated with adavosertib. Error bars represent SEM; performed in duplicate. Crystal violet assay of NCC_CDS1_X1_C1 ( B ) and NCC_CDS2_C1 ( C ) cells comparing adavosertib (IC 50 dose) and DMSO control. ( D ) Immunoblot analysis of NCC_CDS1_X1_C1 cells treated with adavosertib or DMSO control. Representative figure; performed in duplicate. Lysates were run on parallel gels. FL, full-length; Cl, cleaved. ( E ) Immunoblot analysis of NCC_CDS2_C1 cells treated with adavosertib or DMSO control. Representative figure; performed in duplicate. ( F and G ) Relative caspase-3/7 activity in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells treated with adavosertib versus control. One-way ANOVA; performed in triplicate. ( H and I ) Immunoblot analysis of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing 2 independent WEE1 siRNAs. Representative figure; performed in duplicate. ( J and K ) CTG viability assay comparing 2 independent WEE1 siRNAs with scramble control in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. One-way ANOVA; performed in triplicate. ( L and M ) Viability assay comparing 2 independent WEE1 siRNAs with scramble control (siScrm) in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells. One-way ANOVA; performed in duplicate. ( N and O ) Adavosertib IC 50 dose in NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing siCDK1, siCDK2, or siCDK1 and siCDK2. One-way ANOVA; performed in triplicate. ( P ) Relative viability comparing NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells expressing CDK1 WT or CDK1 AF versus vector control. One-way ANOVA. Error bars represent SEM; performed in triplicate. ( Q ) Adavosertib IC 50 dose in NCC_CDS2_C1 cells expressing siCCNE1 or siCtrl. Performed in triplicate. * P <0.001, Student’s t test. ( R ) Adavosertib IC 50 dose in NCC_CDS1_X1_C1 cells expressing si CCNE1 , si CCNE2 , si CCNE1 , and si CCNE2 compared with siCtrl; performed in triplicate. * P <0.001, 1-way ANOVA.

    Article Snippet: Antibodies to PARP, phosphorylated (Y15) CDK1, HSP90, and total CDK1 were purchased from CST.

    Techniques: Viability Assay, Crystal Violet Assay, Western Blot, Activity Assay, Expressing, Plasmid Preparation

    (A) Immunoblot analysis for the G2-M cell cycle markers: pCDC2(T15), total-CDC2, and cyclin B1 on the Icmt +/+ (WT) and Icmt −/− (N1, N2, and N3) isogenic cell lines harvested after growing under standard culture conditions before reach confluency and processed for SDS–PAGE. The numbers below each marker are the densitometry quantification of band intensity. (B) Cell cycle distribution of the isogenic cells in the baseline state without synchronization (top row), and following double thymidine synchronization (0 h, middle row) and 8 h post-release (bottom row). The red vertical lines mark the G1 and G2/M peaks. The cells were prepared by standard propidium iodide staining method for cell cycle analysis, as described in the Materials and Methods section. (C) Flow cytometry analysis of Citrine-Geminin and mCherry-Cdt1 in the isogenic cell lines stably expressing these cell cycle markers. Analysis was performed at the baseline state without synchronization (top row), at the time of release from the double thymidine block (middle row, 0 h), and 8 h after release from double thymidine block (bottom row). (B) These cells were subjected to the same preparation conditions as in (B) before standard flow cytometry analysis. The experiments were performed three times with similar findings.

    Journal: Life Science Alliance

    Article Title: Suppression of isoprenylcysteine carboxylmethyltransferase compromises DNA damage repair

    doi: 10.26508/lsa.202101144

    Figure Lengend Snippet: (A) Immunoblot analysis for the G2-M cell cycle markers: pCDC2(T15), total-CDC2, and cyclin B1 on the Icmt +/+ (WT) and Icmt −/− (N1, N2, and N3) isogenic cell lines harvested after growing under standard culture conditions before reach confluency and processed for SDS–PAGE. The numbers below each marker are the densitometry quantification of band intensity. (B) Cell cycle distribution of the isogenic cells in the baseline state without synchronization (top row), and following double thymidine synchronization (0 h, middle row) and 8 h post-release (bottom row). The red vertical lines mark the G1 and G2/M peaks. The cells were prepared by standard propidium iodide staining method for cell cycle analysis, as described in the Materials and Methods section. (C) Flow cytometry analysis of Citrine-Geminin and mCherry-Cdt1 in the isogenic cell lines stably expressing these cell cycle markers. Analysis was performed at the baseline state without synchronization (top row), at the time of release from the double thymidine block (middle row, 0 h), and 8 h after release from double thymidine block (bottom row). (B) These cells were subjected to the same preparation conditions as in (B) before standard flow cytometry analysis. The experiments were performed three times with similar findings.

    Article Snippet: All antibodies, including those for β-actin, pMEK (#9121), MEK, pERK (#4377), ERK, cleaved Cas7 (#8438), p-γH2AX (#9718), pCDC2 (#4539), total-CDC2 (#28439), cyclin B1 (#4135), GAPDH (#5174), phospo-DNAPKs (2056), cyclin D1 (#2922), pAKT (#9271S), and total AKT (#9272S) were purchased from Cell Signaling Technology.

    Techniques: Western Blot, SDS Page, Marker, Staining, Cell Cycle Assay, Flow Cytometry, Stable Transfection, Expressing, Blocking Assay