Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A ) In vitro CDK1 activity (top2 plots) was analyzed in EOC cells with or without ODF2L knockdown and then treated with DMSO or AZD1775 (treatment: DMSO/200 nM AZD1775, 4 hours). ( B ) Immunoblots of the T14 and Y15 phosphorylation status of CDK1 in the indicated EOC cells (treatment: DMSO/200 nM AZD1775, 24 hours). ( C and D ) In vitro CDK1 activity (top 2 plots) was analyzed in CDK1 T14A–expressing ( C ), CDK1 Y15F–expressing ( D ), and CDK1 WT–expressing ( C and D ) EOC cells with endogenous CDK1 removed (treatment: DMSO or 200 nM AZD1775 for 4 hours). ( E and F ) Representative images and quantification of colony formation ( E ) and apoptotic cell death ( F ) of EOC cells with or without ODF2L knockdown. Cells were treated with sublethal doses of AZD1775 (A2780, 200 nM; SKOV3, 200 nM) together with or without 5 μM Ro-3306 for 72 hours. Data are the mean ± SD from 3 technical replicates of each sample and are representative of 3 ( A ), 2 ( B ), and 3 ( C – F ) independent biological experiments. Blots shown were run in parallel, contemporaneously using the same cell lysate harvested from 1 representative experiment for each cell line in A – D . *** P < 0.001 and **** P < 0.0001, by 1-way ANOVA.

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: In Vitro, Activity Assay, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A ) Analysis of correlations between CDK1 expression levels and those of WEE1, PKMYT1, and ODF2L using TCGA OV data. ( B ) Analysis of the endogenous interaction between ODF2L and PKMYT1 by reverse coimmunoprecipitation followed by immunoblotting. ( C and D ) Analysis of the interaction between CDK1 and PKMYT1 in ODF2L-knockdown EOC cells ( C ) and the interaction between CDK1 and ODF2L in PKMYT1 knockdown EOC cells ( D ) by immunoprecipitation of endogenous CDK1 followed by immunoblotting. ( E ) BRET assay for the binding of PKMYT1 and CDK1 (plasmid design strategy is shown on left) measured in the indicated cells transfected with Halo-tagged CDK1 and Nluc-tagged PKMYT1 in the absence or presence of ODF2L. ( F ) Analysis of the binding of CDK1 and PKMYT1 to the C-terminally, middle-range, and N-terminally truncated ODF2L by coimmunoprecipitation. The indicated cells were cotransfected with Myc-PKMYT1, CDK1-V5, or Flag-tagged truncated ODF2L domains. ( G and H ) Analysis of cell viability upon AZD1775 treatment with or without ODF2L knockdown upon overexpression of PKMYT1 ( G ) or knockdown of PKMYT1 ( H ). AZD1775 (A2780, 200 nM; SKOV3, 200 nM). Data are the mean ± SD from 3 technical replicates in E , G , and H . Data are representative of 2 ( B ), 2 ( C ), 2 ( D ), 3 ( E ), 2 ( F ), 2 ( G ), and 2 ( H ) independent biological experiments. Blots shown were run in parallel, contemporaneously using the same cell lysate harvested from 1 representative experiment for each cell line in H . ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 1-way ANOVA.

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Immunoprecipitation, Bioluminescence Resonance Energy Transfer, Binding Assay, Plasmid Preparation, Transfection, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A and B ) In vitro CDK1 activity, total ODF2L expression levels ( A ) and cell viability ( B ) were analyzed in AZD1775-treated EOC cells derived from the primary tumor tissue of 57 ovarian cancer patients. Patient 1 was used as a control for different batches and labeled blue. ( C and D ) Analysis of correlations between ODF2L expression levels and CDK1 activity ( C ) or cell viability ( D ) in AZD1775-treated primary EOC cells. ODF2L expression levels and CDK1 activity were measured by quantification of the blots in A using ImageJ software. ( E ) Effect of ODF2L expression levels on in vivo tumor growth of PDXs treated with AZD1775. Tumor tissue from patients 1, 5, and 10; patients 3 and 21; and patients 33 and 36 were chosen for the xenograft on the basis of the differential expression levels of ODF2L in the primary cells confirmed by Western blotting. Mice were evenly grouped when the volume of their tumors reached approximately 100 mm 3 25 days after xenografting and were treated with vehicle or AZD1775 (40 mg/kg, orally, once per day). ID, identification. Data are representative of 2 ( A ) and 3 ( B ) independent biological experiments and represent the mean ± SD of 3 technical replicates of each sample ( B ). Error bars in E represent the SEM for tumor volume ( n = 6). ** P < 0.01 and *** P < 0.0001, by 2-tailed Pearson’s correlation coefficient ( C and D ), 2-way ANOVA for tumor volume ( E ), and 1-way ANOVA for tumor weight ( F ).

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: In Vitro, Activity Assay, Expressing, Derivative Assay, Labeling, Software, In Vivo, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A ) Schematic illustration of targeted LNP against ovarian ID8 cells using the ASSET platform (upper panel) and experimental design (lower panel). ( B and C ) Effect of combination treatment with siODF2L-LNP and AZD1775 on ID8 tumor growth. In vivo bioluminescence image ( B ) and average photonic flux ( C ) at week 7. LNPs (0.75 mg/kg, twice a week); AZD1775 (30 mg/kg, orally, once per day). n = 10. ( D ) Survival curves of ID8-bearing mice in the indicated groups. n = 10. ( E ) Quantification of peritoneal ID8 tumor nodule numbers in mice at the endpoint. ( F and G ) In vitro CDK1 activity and total ODF2L expression levels were analyzed by Western blotting ( F ), and the level of the DNA damage marker γH2AX was analyzed ( G ) by flow cytometry in the 2 or 3 ID8 tumors harvested from mice in each group at the endpoint. Data represent the mean ± SD; n = 10 ( B – E ). Data are the mean ± SD; n = 2 or n = 3 ( G ). Data are representative of 2 ( F ) and 3 ( G ) independent biological experiments. ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 1-way ANOVA.

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: In Vivo, In Vitro, Activity Assay, Expressing, Western Blot, Marker, Flow Cytometry

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

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

Journal: PLoS ONE

Article Title: Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

doi: 10.1371/journal.pone.0028602

Figure Lengend Snippet: (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the Cdk1 inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).

Article Snippet: Samples were resolved in 7.5% or 15% SDS-PAGE, blotted to Immobilon-P membranes (Millipore) and probed with the following antibodies: monoclonal anti-AIB1 (BD Biosciences), polyclonal anti-phospho-AKT Ser473, polyclonal anti-phospho-Cdc2 Tyr15, polyclonal anti-phospho-PP1 Thr320 and polyclonal anti-Cdc2 (Cell Signaling); monoclonal anti-phospho-ERK1/2 Tyr204, polyclonal anti-cyclin A, polyclonal anti-cyclin B1, monoclonal anti-cyclin D1, polyclonal anti-cyclin E, polyclonal anti-PP1, monoclonal anti-Lamin A/C from (Santa Cruz Biotech); monoclonal anti-phospho-histone H3 from Millipore; monoclonal anti-β-actin from Sigma and monoclonal anti-ubiquitin FK2 (Affinity Bioreagents).

Techniques: Western Blot, Activity Assay, Immunoprecipitation, Produced, Recombinant, Purification, Affinity Chromatography, Incubation, SDS Page, Positive Control

Journal: PLoS ONE

Article Title: Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

doi: 10.1371/journal.pone.0028602

Figure Lengend Snippet: Floating HeLa cells arrested at mitosis with nocodazole were further treated for three more hours with vehicle alone as control (lane 1), 10 µM purvalanol A (lane 2), 1.25 µM Aurora kinase inhibitor II (lane 3), 7 µM Plk inhibitor III (lane 4) or 100 µM of the Hec1/Nek2 inhibitor I (lane 5). Cells were lysed and analyzed by westernblotting against the indicated molecular species. Importantly, exposure of cells to the Cdk1 inhibitor caused the desphosphorylation of AIB1 together with a rapid mitotic exit (lane 2) to a similar extent as previously reported with other inhibitors , . On the other hand, exposure of mitotic cells to inhibitors specific for Aurora A, B, Plks or Nek2 did not show any significant effect on AIB1 phosphorylation, suggesting that neither of them is a kinase for AIB1 under the experimental conditions tested. The protein kinase inhibitor staurosporine causes morphological changes at mitosis in HeLa cells, including the decondensation of chromosomes and the reformation of nuclear membrane . Treatment with the different kinase inhibitors at the concentrations indicated by the supplier caused similar morphological changes plus the reattachment of cells to the surface of the petri dish (data not shown), suggesting that the inhibitors indeed exerted a biologically relevant activity.

Article Snippet: Samples were resolved in 7.5% or 15% SDS-PAGE, blotted to Immobilon-P membranes (Millipore) and probed with the following antibodies: monoclonal anti-AIB1 (BD Biosciences), polyclonal anti-phospho-AKT Ser473, polyclonal anti-phospho-Cdc2 Tyr15, polyclonal anti-phospho-PP1 Thr320 and polyclonal anti-Cdc2 (Cell Signaling); monoclonal anti-phospho-ERK1/2 Tyr204, polyclonal anti-cyclin A, polyclonal anti-cyclin B1, monoclonal anti-cyclin D1, polyclonal anti-cyclin E, polyclonal anti-PP1, monoclonal anti-Lamin A/C from (Santa Cruz Biotech); monoclonal anti-phospho-histone H3 from Millipore; monoclonal anti-β-actin from Sigma and monoclonal anti-ubiquitin FK2 (Affinity Bioreagents).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

doi: 10.1371/journal.pone.0028602

Figure Lengend Snippet: (A) Representative scheme of full length AIB1 and GST fusion fragments generated and named A through E. (B) Qualitative analysis of AIB1 fragments subjected to in vitro phosphorylation with active Cdk1/cyclin B. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fussion protein and 1 µg was used in a parallel reaction as a positive control. (C) Same fragments as in (B) were also incubated with 100 ng of active Cdk1/Cyclin A2 (Cell Signaling). (D) Coomassie staining of the different AIB1 fragments fused to GST. Based in the cualitative amounts of proteins stained, double input of fragment E was used in the radioactive reactions. (E) Autoradiography (upper panel) of AIB1 fragment C (aminoacids 693–933) and fragments harbouring point substitutions serine 728 to alanine (S728A), serine 860 to alanine (S860A) and serine 867 to alanine (S867A), subjected to phosphorylation by complex Cdk1/cyclin B1 in the presence of [γ- 32 P]ATP, 1 hour at 30°C. Bottom panel represents a Coomassie staining of the fragments used in the upper kinase reactions. (F) Autoradiography exposure (upper panel) and coomassie staining (lower panel) of AIB1 fragment C and a mutant (C AYA) in which amino acids RYL localized at +11 from serine 728 are mutated to AYA. Fragments were incubated with active Cdk1/cyclin B1 for 1 hour at 30°C in the presence of [γ- 32 P]ATP. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fusion protein and used in a parallel reaction as a positive control.

Article Snippet: Samples were resolved in 7.5% or 15% SDS-PAGE, blotted to Immobilon-P membranes (Millipore) and probed with the following antibodies: monoclonal anti-AIB1 (BD Biosciences), polyclonal anti-phospho-AKT Ser473, polyclonal anti-phospho-Cdc2 Tyr15, polyclonal anti-phospho-PP1 Thr320 and polyclonal anti-Cdc2 (Cell Signaling); monoclonal anti-phospho-ERK1/2 Tyr204, polyclonal anti-cyclin A, polyclonal anti-cyclin B1, monoclonal anti-cyclin D1, polyclonal anti-cyclin E, polyclonal anti-PP1, monoclonal anti-Lamin A/C from (Santa Cruz Biotech); monoclonal anti-phospho-histone H3 from Millipore; monoclonal anti-β-actin from Sigma and monoclonal anti-ubiquitin FK2 (Affinity Bioreagents).

Techniques: Generated, In Vitro, SDS Page, Autoradiography, Positive Control, Incubation, Staining, Mutagenesis

Journal: PLoS ONE

Article Title: Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

doi: 10.1371/journal.pone.0028602

Figure Lengend Snippet: (A) Western blot analysis of 30 µg of cleared lysates from nocodazole-arrested HeLa cell which were treated with the indicated phosphatases for 1.5 h at 30°C. Phospho-Akt antibodies were used as a control of the enzymatic activity. (B) Cells arrested at mitosis with nocodazole were further treated with vehicle alone (−) or with the Cdk1 inhibitor purvalanol A 10 mM (+) during 3 hours. Anti-lamin A/C antibodies were used to control loading. (C) Mitotic cells were treated with the MEK inhibitor PD98059 20 µM (+) or with vehicle alone (−) and cell lysates were analyzed by western blotting with AIB1 antibodies. Phospho-ERK specific antibodies were used as control for the efficiency of the treatment. (D) Western blot analysis of immunoprecipitated complexes using anti-AIB1 antibodies or pre-immune serum (Pre-I) from lysates of asynchronous HeLa cells (A) or from cells arrested with nocodazole (M). (E) Human AIB1 was subcloned in pFASTBac HTa and baculovirus were produced to express the recombinant full-length His6-AIB1 in Sf9 cells using the system BacPak (Clontech). Recombinant protein was purified by standard Ni 2+ -NTA affinity chromatography followed by desalting with a molecular exclusion column. Purified AIB1 (middle panel) was incubated with [γ- 32 P]ATP in the presence (+) or absence (−) of recombinant Cdk1/cyclin B1 for 1 hour at 30°C (upper panel). Reactions were resolved by SDS-PAGE, the gel was dryed and exposed to an X-ray film. 1 µg of histone H1 (Sigma) was used as a positive control (bottom panel).

Article Snippet: Samples were resolved in 7.5% or 15% SDS-PAGE, blotted to Immobilon-P membranes (Millipore) and probed with the following antibodies: monoclonal anti-AIB1 (BD Biosciences), polyclonal anti-phospho-AKT Ser473, polyclonal anti-phospho-Cdc2 Tyr15, polyclonal anti-phospho-PP1 Thr320 and polyclonal anti-Cdc2 (Cell Signaling); monoclonal anti-phospho-ERK1/2 Tyr204, polyclonal anti-cyclin A, polyclonal anti-cyclin B1, monoclonal anti-cyclin D1, polyclonal anti-cyclin E, polyclonal anti-PP1, monoclonal anti-Lamin A/C from (Santa Cruz Biotech); monoclonal anti-phospho-histone H3 from Millipore; monoclonal anti-β-actin from Sigma and monoclonal anti-ubiquitin FK2 (Affinity Bioreagents).

Techniques: Western Blot, Activity Assay, Immunoprecipitation, Produced, Recombinant, Purification, Affinity Chromatography, Incubation, SDS Page, Positive Control

Journal: PLoS ONE

Article Title: Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

doi: 10.1371/journal.pone.0028602

Figure Lengend Snippet: Floating HeLa cells arrested at mitosis with nocodazole were further treated for three more hours with vehicle alone as control (lane 1), 10 µM purvalanol A (lane 2), 1.25 µM Aurora kinase inhibitor II (lane 3), 7 µM Plk inhibitor III (lane 4) or 100 µM of the Hec1/Nek2 inhibitor I (lane 5). Cells were lysed and analyzed by westernblotting against the indicated molecular species. Importantly, exposure of cells to the Cdk1 inhibitor caused the desphosphorylation of AIB1 together with a rapid mitotic exit (lane 2) to a similar extent as previously reported with other inhibitors , . On the other hand, exposure of mitotic cells to inhibitors specific for Aurora A, B, Plks or Nek2 did not show any significant effect on AIB1 phosphorylation, suggesting that neither of them is a kinase for AIB1 under the experimental conditions tested. The protein kinase inhibitor staurosporine causes morphological changes at mitosis in HeLa cells, including the decondensation of chromosomes and the reformation of nuclear membrane . Treatment with the different kinase inhibitors at the concentrations indicated by the supplier caused similar morphological changes plus the reattachment of cells to the surface of the petri dish (data not shown), suggesting that the inhibitors indeed exerted a biologically relevant activity.

Article Snippet: Samples were resolved in 7.5% or 15% SDS-PAGE, blotted to Immobilon-P membranes (Millipore) and probed with the following antibodies: monoclonal anti-AIB1 (BD Biosciences), polyclonal anti-phospho-AKT Ser473, polyclonal anti-phospho-Cdc2 Tyr15, polyclonal anti-phospho-PP1 Thr320 and polyclonal anti-Cdc2 (Cell Signaling); monoclonal anti-phospho-ERK1/2 Tyr204, polyclonal anti-cyclin A, polyclonal anti-cyclin B1, monoclonal anti-cyclin D1, polyclonal anti-cyclin E, polyclonal anti-PP1, monoclonal anti-Lamin A/C from (Santa Cruz Biotech); monoclonal anti-phospho-histone H3 from Millipore; monoclonal anti-β-actin from Sigma and monoclonal anti-ubiquitin FK2 (Affinity Bioreagents).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

doi: 10.1371/journal.pone.0028602

Figure Lengend Snippet: (A) Representative scheme of full length AIB1 and GST fusion fragments generated and named A through E. (B) Qualitative analysis of AIB1 fragments subjected to in vitro phosphorylation with active Cdk1/cyclin B. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fussion protein and 1 µg was used in a parallel reaction as a positive control. (C) Same fragments as in (B) were also incubated with 100 ng of active Cdk1/Cyclin A2 (Cell Signaling). (D) Coomassie staining of the different AIB1 fragments fused to GST. Based in the cualitative amounts of proteins stained, double input of fragment E was used in the radioactive reactions. (E) Autoradiography (upper panel) of AIB1 fragment C (aminoacids 693–933) and fragments harbouring point substitutions serine 728 to alanine (S728A), serine 860 to alanine (S860A) and serine 867 to alanine (S867A), subjected to phosphorylation by complex Cdk1/cyclin B1 in the presence of [γ- 32 P]ATP, 1 hour at 30°C. Bottom panel represents a Coomassie staining of the fragments used in the upper kinase reactions. (F) Autoradiography exposure (upper panel) and coomassie staining (lower panel) of AIB1 fragment C and a mutant (C AYA) in which amino acids RYL localized at +11 from serine 728 are mutated to AYA. Fragments were incubated with active Cdk1/cyclin B1 for 1 hour at 30°C in the presence of [γ- 32 P]ATP. Reactions were resolved by SDS-PAGE and gel, dried on Whatman paper and exposed for autoradiography. Fragment containing amino acids 792–928 of the Retinoblastoma protein (Rb) was also expressed as a GST fusion protein and used in a parallel reaction as a positive control.

Article Snippet: Samples were resolved in 7.5% or 15% SDS-PAGE, blotted to Immobilon-P membranes (Millipore) and probed with the following antibodies: monoclonal anti-AIB1 (BD Biosciences), polyclonal anti-phospho-AKT Ser473, polyclonal anti-phospho-Cdc2 Tyr15, polyclonal anti-phospho-PP1 Thr320 and polyclonal anti-Cdc2 (Cell Signaling); monoclonal anti-phospho-ERK1/2 Tyr204, polyclonal anti-cyclin A, polyclonal anti-cyclin B1, monoclonal anti-cyclin D1, polyclonal anti-cyclin E, polyclonal anti-PP1, monoclonal anti-Lamin A/C from (Santa Cruz Biotech); monoclonal anti-phospho-histone H3 from Millipore; monoclonal anti-β-actin from Sigma and monoclonal anti-ubiquitin FK2 (Affinity Bioreagents).

Techniques: Generated, In Vitro, SDS Page, Autoradiography, Positive Control, Incubation, Staining, Mutagenesis

Journal: PLoS ONE

Article Title: c-Rel Deficiency Increases Caspase-4 Expression and Leads to ER Stress and Necrosis in EBV-Transformed Cells

doi: 10.1371/journal.pone.0025467

Figure Lengend Snippet: ( A ) Pt1- (dashed line) and control D11-LCL tet cells (solid line) grown in the absence of Tc for 5 days were plated at 1×10 6 cells/mL in Tc media and cultured in parallel for 6 days. Cells were withdrawn at days 1, 3 and 6 cells and counted using Trypan-Blue exclusion to determine the total number of viable cells. ( B ) 1×10 6 Pt1- and control D11- and C2-LCL tet cells were stained with CFSE and cultured in Tc media (pink line), absence of Tc (for 5 days prior to CFSE staining) (grey peak) or absence of Tc plus sCD40L (500 ng/mL) (green line) for 6 days. At day 4 (upper panel) and day 6 (lower panel) cells were analyzed for proliferation by flow cytometry. ( C ) Pt1- and control D11-LCL tet cells (2×10 5 ) grown in Tc media were analyzed for the expression of phosphorylated-CDC2 by intra-cellular staining. Numbers shown are the mean fluorescence intensity (MFI) of cells expressing high levels of phosphorylated CDC2. Graph is representative of three independent experiments.

Article Snippet: Anti-S6 mAb, rabbit polyclonal anti-caspase 4 and rabbit polyclonal anti-phospho-CDC2 were purchased from Cell Signaling.

Techniques: Cell Culture, Staining, Flow Cytometry, Expressing, Fluorescence

Journal: Molecular Neurodegeneration

Article Title: Mitosis-specific phosphorylation of amyloid precursor protein at Threonine 668 leads to its altered processing and association with centrosomes

doi: 10.1186/1750-1326-6-80

Figure Lengend Snippet: Increased expression of cell cycle regulatory proteins in AD transgenic mice : A) Cyclin E, E2F1 and P-cdc2 levels are upregulated in transgenic mice expressing APP and PS/APP: Brain sections from Ntg normal mice (a, c, f) were compared to those from transgenic mice expressing APP (d, g) and PS/APP (b, e, h) using cyclin E (upper panel), E2F1 (middle panel) or P-cdc2 (lower panel) antibodies. Images (a-e, 5× and f-h 20×) were taken using a Nikon E1000 microscope and analyzed using Image-Pro Plus software. The P-cdc2 and images in the inset show magnified images (20×) of the plaques to visualize the cells. We found that the cells surrounding the plaques were positive for cyclin E, E2F1, and P-cdc2, and it appears that both neurons (black arrow head) and glia (white arrow head) were positive for P-cdc2. 'Secondary antibodies only' control did not show any specific staining of the sections (data not shown). B) Quantitative analysis of cyclin D1 and E expression in APP and PS/APP transgenic mice brains: Brain sections from Ntg and mice expressing APP and PS/APP were stained using a monoclonal cyclin D1 or a polyclonal cyclin E antibody and nuclei visualized using Hoechst. The signal intensity was measured using Image J, image processing and analysis program. The signal strength was compared to that with Hoechst nuclear staining from each section to avoid mouse-to-mouse variation. The means of results from six independent mice are shown with standard error bars and P values. While APP mice showed a significantly higher level of only cyclin E compared to cyclin D1, PS/APP mice showed higher levels of both cyclin D1 and E levels compared to Ntg.

Article Snippet: Anti-Aβ/APP antibody (6E10 raised against Aβ1-16) was from Signet, C-terminal APP antibody was from Chemicon/Millipore, Thr668 P-APP, MPM-2, and P-cdc2 antibodies were from Cell Signaling, and cyclin D1, cyclin E, and E2F1 antibodies were from Santa Cruz Biotechnology.

Techniques: Expressing, Transgenic Assay, Microscopy, Software, Staining