Journal: Cell reports

Article Title: Elevating PLK1 overcomes BETi resistance in prostate cancer via triggering BRD4 phosphorylation-dependent degradation in mitosis

doi: 10.1016/j.celrep.2024.114431

Figure Lengend Snippet: (A) IF staining of C4-2 cells to detect BRD4 (red) and PLK1 (green). The white broken circles indicate mitotic cells. (B) WB analysis of PCa cells treated with DMSO, nocodazole (Noc.), or double-thymidine block (DTB). (C) WB of PCa cells released from nocodazole treatment. (D) WB of C4-2 cells released from DTB treatment. (E) WB of C4-2 cells upon indicated treatment.

Article Snippet: Recombinant Human PLK1 Protein (Active) , Sino Biological US Inc , Cat# 10676-H07B.

Techniques: Staining, Blocking Assay

Journal: Cell reports

Article Title: Elevating PLK1 overcomes BETi resistance in prostate cancer via triggering BRD4 phosphorylation-dependent degradation in mitosis

doi: 10.1016/j.celrep.2024.114431

Figure Lengend Snippet: (A) Immunoprecipitation (IP) assay of HEK293T lysates with FLAG-PLK. (B) IP assay of C4-2 cell lysates with anti-BRD4 antibody. (C) The schematic structure of BRD4. (D) IP assay with HA-BRD4 fragments. (E) The schematic structure of PLK1. (F) IP assay using FLAG-PLK1 domains. (G) Proximity ligation assay (PLA) of BRD4 and PLK1 (left). Scale bar, 50 μm. The representative PLA images of metaphase cells are highlighted in the middle panel. The right histogram shows the quantified PLA signal as means ± SD of cells at M phase and at non-M phase. *** p < 0.001. (H) WB analysis of prostate non-transformed or cancer cell lines. The histogram on the right shows quantified BRD4 and PLK1. (I) The correlation analysis of BRD4 and PLK1 mRNA in PCa cell lines (GSE21032). (J) WB analysis of RWPE1 or C4-2/tet-inducible-PLK1 cells treated with doxycycline. (K) WB analysis of DU145/tet-shPLK1 cells treated with the indicated dosage of doxycycline. (L) WB of HEK293T cells transfected indicated plasmids upon cycloheximide treatment.

Article Snippet: Recombinant Human PLK1 Protein (Active) , Sino Biological US Inc , Cat# 10676-H07B.

Techniques: Immunoprecipitation, Proximity Ligation Assay, Transformation Assay, Transfection

Journal: Cell reports

Article Title: Elevating PLK1 overcomes BETi resistance in prostate cancer via triggering BRD4 phosphorylation-dependent degradation in mitosis

doi: 10.1016/j.celrep.2024.114431

Figure Lengend Snippet: (A) The left panel displays the schematic structure of GST-BRD4 fragments, while the right panel illustrates the result of an in vitro kinase assay. (B and C) In vitro kinase assays of purified GST-BRD4 WT or indicated alanine mutants. The upper panels display the conserved sequences of BRD4 among different species. (D) WB analysis of HEK293T cells transfected with plasmids indicated. The right panel indicates the quantified HA-BRD4 abundance. (E) WB of HEK293T cells transfected with plasmids indicated upon cycloheximide (CHX) treatment. (F) WB of C4-2 cells treated with thymidine (Thy.), nocodazole (Noc.), or DMSO (Asyn.). (G) WB of LNCaP (upper panel) and C4-2 (lower panel) cells released from nocodazole treatment. (H) WB of C4-2/tet-inducible PLK1 cells treated with increasing dosages of doxycycline. (I) WB of DU145/tet-shPLK1 cells upon doxycycline induction (left) or 22Rv1 cells upon PLK1 activity inhibition (right). (J) Representative images of IHC in human PCa samples. The red arrow indicates the same cell in serial slides. (K) Representative images of IHC against PLK1 or BRD4 in a TMA of PCa patients. (L) The correlation analysis between PLK1 and BRD4 based on the scores of (K). (M) The trend of PLK1 and BRD4 protein between adenocarcinoma and neuroendocrine PCa patients (ProteomeXchange #PXD042867). (N) The correlation analysis of pT210-PLK1 and pS1100-BRD4 using phosphoproteomic data of breast invasive carcinoma (TCGA, Firehose Legacy).

Article Snippet: Recombinant Human PLK1 Protein (Active) , Sino Biological US Inc , Cat# 10676-H07B.

Techniques: In Vitro, Kinase Assay, Purification, Transfection, Activity Assay, Inhibition

Journal: Cell reports

Article Title: Elevating PLK1 overcomes BETi resistance in prostate cancer via triggering BRD4 phosphorylation-dependent degradation in mitosis

doi: 10.1016/j.celrep.2024.114431

Figure Lengend Snippet: (A) WB of HEK293T cells transfected with HA-BRD4 and FLAG-PLK1 upon treatment of MG132, 3-MA, or chloroquine (CQ). (B) WB of C4-2 cells treated with nocodazole and MG132 in different manners. (C) Ubiquitination assay of HA-BRD4 with co-expression of FLAG-PLK1 and Myc-Ub. (D) The upper panel illustrates the APC/C complex working model during the cell cycle, while the lower panel shows WB analysis of HEK293T cells transfected with indicated plasmids. (E) WB of HEK293T cells transfected indicated plasmids, with the lower panel showing the relative HA-BRD4 amount. (F) WB of C4-2/tet-PLK1 cells transfected with indicated small interfering RNAs (siRNAs), with the lower panel showing the relative levels of endogenous BRD4. (G) Ubiquitination of HA-BRD4 with co-expression of indicated plasmids. (H) IP assay of HEK293T cells lysates transfected with indicated plasmids. (I) IP assay of Myc-CDH1 and HA-BRD4s. (J) Ubiquitination assay of HA-BRD4s with or without Myc-CDH1. (K) WB of HEK293T cells transfected with indicated plasmids upon CHX treatment, with the relative protein amounts of HA-BRD4s at each time point shown.

Article Snippet: Recombinant Human PLK1 Protein (Active) , Sino Biological US Inc , Cat# 10676-H07B.

Techniques: Transfection, Ubiquitin Assay, Expressing

Journal: Cell reports

Article Title: Elevating PLK1 overcomes BETi resistance in prostate cancer via triggering BRD4 phosphorylation-dependent degradation in mitosis

doi: 10.1016/j.celrep.2024.114431

Figure Lengend Snippet: (A) WB analysis of HEK293T cells transfected with indicated plasmids. The right panel shows the relative HA-BRD4 amount. (B) WB of C4-2/HA-SPOP-F133V and FLAG-PLK1 cells, with the right panel showing the relative endogenous BRD4. (C) The cell viability of C4-2/tet-PLK1 upon doxycycline and dosage of JQ1 treatment. ** p < 0.01. (D) Representative colony images, with the quantity of each group on the right panel. (E) WB analysis of C4-2/tet-PLK1 and SPOP-W131G or control cells upon dosages of JQ1 and doxycycline treatment, with the right panel showing the relative levels of c-PARP.

Article Snippet: Recombinant Human PLK1 Protein (Active) , Sino Biological US Inc , Cat# 10676-H07B.

Techniques: Transfection, Control

Journal: Cell reports

Article Title: Elevating PLK1 overcomes BETi resistance in prostate cancer via triggering BRD4 phosphorylation-dependent degradation in mitosis

doi: 10.1016/j.celrep.2024.114431

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant Human PLK1 Protein (Active) , Sino Biological US Inc , Cat# 10676-H07B.

Techniques: Virus, Plasmid Preparation, Recombinant, Magnetic Beads, Mutagenesis, Proximity Ligation Assay, Reverse Transcription, SYBR Green Assay, Software

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Pericentromeric cohesin is deprotected long before sister chromatids separate. During phosphorylation- and Wapl-dependent release of cohesin from chromosome arms, pericentromeric cohesin is protected by associated Sgo1-PP2A. Following the early mitotic degradation of Nek2a and cyclin A2, Sgo1-PP2A relocates to kinetochores. Yet, pericentromeric cohesion persists until securin and cyclin B1 are degraded and separase is activated. A requirement for Nek2a and cyclin A2 for prophase pathway signaling would explain why, in metaphase, Wapl can no longer release cohesin. ( B ) Nek2a and Cdk1/2-cyclin A2 are necessary for Wapl-dependent in vitro displacement of cohesin from chromatin. Chromatin was freshly purified by repeated sedimentation from G2-arrested HeLaK cells, incubated with recombinant Wapl, kinases, and inhibitors (BI2536, ZM-447439, and RO-3306), as indicated, and then re-pelleted. Supernatants and chromatin pellets were analyzed by immunoblotting. ( C ) Wapl and the three kinases Nek2a, Cdk1/2-cyclin A2, and Aurora B-INCENP are sufficient to support the displacement of cohesin from isolated chromatin. Chromatin was freshly purified by repeated sedimentation from G2-arrested HeLaK cells, incubated with recombinant Wapl, Nek2a, Cdk1/2-cyclin A2, Cdk1-cyclin B1, aurora B, and/or Plk1, as indicated, and then re-pelleted. Supernatants and chromatin pellets were analyzed by immunoblotting. .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: In Vitro, Purification, Sedimentation, Incubation, Recombinant, Western Blot, Isolation

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Recombinant kinases are active as judged by the phosphorylation of model substrates. Plk1, aurora B-INCENP, Nek2a, Cdk1/2-cyclin A2, or Cdk1-cyclin B1 supplemented with their specific inhibitor or carrier solvent DMSO (−) were incubated with the corresponding model substrate in the presence of [γ 33 P]-ATP. Reactions were subjected to SDS-PAGE followed by autoradiography. BI BI2536, ZM ZM-447439, ST staurosporine, RO RO-3306, MBP myelin basic protein. ( B ) Recombinant Nek2a, Cdk1/2-cyclin A2, and Wapl are free of cyclin B1 and Plk1. The preparations of Nek2a, Cdk1/2-cyclin A2, Wapl, Cdk1-cyclin B1, and Plk1 used for the release assays were characterized by immunoblotting using the indicated antibodies. In vitro expressed (IVTT) cyclin B1 served as an additional control.

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Recombinant, Solvent, Incubation, SDS Page, Autoradiography, Western Blot, In Vitro, Control

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Experimental outline. DNA-mediated chromatin pull-down from G2-arrested HeLaK cells is followed by cohesin eviction experiments. ( B ) Cohesin is eluted from immobilized chromatin upon DNA-cleavage but not high salt treatment. Chromatin beads were treated with benzonase and/or high salt, and retained proteins were detected by immunoblotting. High-salt-washed chromatin (lane 6) was used for subsequent cohesin eviction experiments. ( C ) Wapl-dependent cohesin release in vitro requires Nek2a, Cdk1/2-cyclin A2, and Aurora B but not Cdk1-cyclin B1 and Plk1. Immobilized chromatin was combined with the indicated proteins. Following centrifugation, DNA-beads and supernatant were analyzed by immunoblotting. .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Western Blot, In Vitro, Centrifugation

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Nek2a and Cdk1/2-cyclin A2 target juxtaposed but distinct residues within the disordered, C-terminal domain of Pds5b. In vitro expressed Pds5b variants were incubated with the indicated kinases and inhibitors in the presence of [γ- 33 P]-ATP, subjected to SDS-PAGE, and analyzed by autoradiography and immunoblotting. Note that Pds5b is also phosphorylated by aurora B and, weakly, by Plk1. KD kinase-dead Nek2a-Lys37Met; IVTT coupled in vitro transcription-translation. ( B ) Graphical summary of Nek2a- and Cdk1/2-cyclin A2-targeted Pds5b residues and corresponding phosphorylation-resistant variants. ( C ) Ser1209 of Pds5b is phosphorylated by Nek2a but not Cdk1/2-cyclin A2. Following incubation of in vitro expressed Pds5b-WT or −3A with Nek2a or Cdk1/2-cyclin A2, samples were analyzed by immunoblotting using the indicated antibodies. Nek2a-Lys37Met (KD, kinase-dead) served as a negative control. ( D ) The spatiotemporal dynamics of Ser1209-phosphorylation correlate with the recruitment of Wapl to chromatin followed by cohesin release. HeLaK cells synchronously cycling through mitosis were subjected to time-resolved fractionation and immunoblotting. At the time of release from a RO-3306-mediated arrest, cultures were supplemented with a Nek2a inhibitor (NCL00017509) or carrier solvent (mock). .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: In Vitro, Incubation, SDS Page, Autoradiography, Western Blot, Negative Control, Fractionation, Solvent

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Recombinant, Transduction, Sequencing, Protease Inhibitor, Membrane, Software

Journal: Cell reports

Article Title: Proteomic analysis reveals a PLK1-dependent G2/M degradation program and a role for AKAP2 in coordinating the mitotic cytoskeleton

doi: 10.1016/j.celrep.2024.114510

Figure Lengend Snippet:

Article Snippet: Recombinant Human Active PLK1 Protein, CF , R&D Systems , 3804-KS.

Techniques: Virus, Recombinant, Infection, Transfection, Western Blot, Saline, Sequencing, Plasmid Preparation, Mutagenesis, Bicinchoninic Acid Protein Assay, Staining, Expressing, Luciferase, Software, Mass Spectrometry

Journal: bioRxiv

Article Title: In vitro reconstitution reveals requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1101/2024.03.12.584565

Figure Lengend Snippet: A Recombinant kinases are active as judged by phosphorylation of model substrates. Plk1, aurora B-INCENP, Nek2a, Cdk1/2-cyclin A2 or Cdk1-cyclin B1 supplemented with their specific inhibitor or carrier solvent DMSO (-) were incubated with corresponding model substrate in presence of [γ 33 P]-ATP. Reactions were subjected to SDS-PAGE followed by autoradiography. BI, BI2536; ZM, ZM447439; ST, staurosporine; RO, RO3306; MBP, myelin basic protein. B Nek2a and Cdk1/2-cyclin A2 are necessary for Wapl-dependent in vitro displacement of cohesin from chromatin. Chromatin was freshly purified by repeated sedimentation from G2-arrested HeLaK cells, incubated with recombinant Wapl, kinases and inhibitors (BI2536, ZM447439 and RO3306), as indicated, and then re-pelleted. Supernatants and chromatin pellets were analyzed by immunoblotting. C Wapl and the three kinases Nek2a, Cdk1/2-cyclin A2 and Aurora B-INCENP are sufficient to support displacement of cohesin from isolated chromatin. Experiment was done as in B).

Article Snippet: Active Plk1 was purchased from R&D Systems (No. 3804-KS).

Techniques: Recombinant, Phospho-proteomics, Solvent, Incubation, SDS Page, Autoradiography, In Vitro, Purification, Sedimentation, Western Blot, Isolation

Journal: bioRxiv

Article Title: In vitro reconstitution reveals requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1101/2024.03.12.584565

Figure Lengend Snippet: A Nek2a and Cdk1/2-cyclin A2 target juxtaposed but distinct residues within the disordered, C-terminal domain of Pds5b. In vitro expressed Pds5b variants were incubated with the indicated kinases and inhibitors in presence of [γ- 33 P]-ATP, subjected to SDS-PAGE and analyzed by autoradiography and immunoblotting. Note that Pds5b is also phosphorylated by aurora B and, weakly, by Plk1. KD = kinase-dead Nek2a-Lys37Met; IVTT = coupled in vitro transcription-translation. B Rendering Pds5b resistant against both Nek2a and Cdk1/2-cyclin A2 is necessary and sufficient to switch it from a Wapl- to a sororin-binder. Shown are the experimental outline and corresponding immunoblots. Note that following the incubation with Nek2a and/or Cdk1/2-cyclin A2, ATP was no longer present to exclude phosphorylation of sororin (and/or Wapl) by traces of kinases, which might be retained on the beads.

Article Snippet: Active Plk1 was purchased from R&D Systems (No. 3804-KS).

Techniques: In Vitro, Incubation, SDS Page, Autoradiography, Western Blot, Phospho-proteomics