Journal: Journal of Cancer

Article Title: CDK1 serves as a novel therapeutic target for endometrioid endometrial cancer

doi: 10.7150/jca.51139

Figure Lengend Snippet: The degree, betweenness and closeness of the 10 hub genes

Article Snippet: CDK1 inhibitor RO3306 (Selleck Chemicals, Houston, USA) was dissolved in DMSO at a concentration of 20 mM and added to the cell culture medium at final concentrations of 0, 2.5, 5, 10, 15 and 20 μM.

Techniques:

Journal: Journal of Cancer

Article Title: CDK1 serves as a novel therapeutic target for endometrioid endometrial cancer

doi: 10.7150/jca.51139

Figure Lengend Snippet: The expression of CDK1 in normal endometrium tissues and endometrioid endometrial cancer tissues. (A) Representative different types of immunostaining for CDK1 in normal endometrium tissues and endometrioid endometrial cancer tissues (original magnification×400). (a) CDK1-negative nuclear and cytoplasmic staining in the majority of normal endometrium tissues. (b) CDK1-positive nuclear staining but CDK1-negative cytoplasmic staining in the minority of normal endometrium tissues. (c) CDK1 strong positive nuclear staining but CDK1 weak positive cytoplasmic staining in the well differentiated endometrioid endometrial cancer tissues. (d) Diffuse strong positive staining for CDK1 in moderately differentiated endometrioid endometrial cancer tissues. (B) The average IHC scores in the nucleus and cytoplasm of normal endometrium tissues (n = 30) were compared with the scores in the nucleus and cytoplasm of cancer tissues (n = 33). Student's independent t-tests was used. Data were presented as means ± SD, ****p <0.0001. (C) Representative immunohistochemical staining for CDK1 in normal endometrium tissues and its matched adjacent cancer tissues. Representative immunostaining for CDK1 in normal endometrium tissues (a) and its matched adjacent cancer tissues (b) of endometrioid endometrial cancer (original magnification×400). (c) A geographic zone of normal endometrium tissues (upper and left) contrasting with matched adjacent region of cancer tissues (down and right half) (original magnification×200). (d) Higher power view of the central region of panel C (original magnification×400). Scale bars 100 µm. (D) The average IHC scores in the nucleus and cytoplasm of normal endometrium tissues (n = 33) were compared with the scores in the nucleus and cytoplasm of matched adjacent cancer tissues (n = 33). Student's paired t-tests was used. Data were presented as means ± SD, ****p <0.0001.

Article Snippet: CDK1 inhibitor RO3306 (Selleck Chemicals, Houston, USA) was dissolved in DMSO at a concentration of 20 mM and added to the cell culture medium at final concentrations of 0, 2.5, 5, 10, 15 and 20 μM.

Techniques: Expressing, Immunostaining, Staining, Immunohistochemical staining

Journal: Journal of Cancer

Article Title: CDK1 serves as a novel therapeutic target for endometrioid endometrial cancer

doi: 10.7150/jca.51139

Figure Lengend Snippet: The CDK1 expression in nucleus and cytoplasm of normal endometrium and endometrial cancer

Article Snippet: CDK1 inhibitor RO3306 (Selleck Chemicals, Houston, USA) was dissolved in DMSO at a concentration of 20 mM and added to the cell culture medium at final concentrations of 0, 2.5, 5, 10, 15 and 20 μM.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: CDK1 serves as a novel therapeutic target for endometrioid endometrial cancer

doi: 10.7150/jca.51139

Figure Lengend Snippet: CDK1 expression and clinicopathological parameters in 33 endometrial cancer patients

Article Snippet: CDK1 inhibitor RO3306 (Selleck Chemicals, Houston, USA) was dissolved in DMSO at a concentration of 20 mM and added to the cell culture medium at final concentrations of 0, 2.5, 5, 10, 15 and 20 μM.

Techniques: Expressing, Staining

Journal: Journal of Cancer

Article Title: CDK1 serves as a novel therapeutic target for endometrioid endometrial cancer

doi: 10.7150/jca.51139

Figure Lengend Snippet: RO3306 as a potent CDK1 inhibitor inhibited the cell growth and colony formation of HEC-1-B cells in vitro . (A, B) HEC-1-B cells were treated with RO3306 at different concentrations of 0, 2.5, 5, 10, 15 and 20 µM. Cell viability was determined at 24, 48, and 72 hours by a CCK-8 assay. Cell viability values are expressed relative to those for cells without drug exposure. Data were presented as means ± SD (n = 3). (A) Time-response effect of RO3306 on HEC-1-B cells. (B) Dose-response effect of RO3306 on HEC-1-B cells. The IC50 value for RO3306 at 72 h were obtained by GraphPad Prism version 7.0 software. (C) Colony formation assay. HEC-1-B cells were treated with RO3306 at different concentrations of 0, 2.5 and 5 µM for 48 h. Cells were fixed and stained at the end of the study.

Article Snippet: CDK1 inhibitor RO3306 (Selleck Chemicals, Houston, USA) was dissolved in DMSO at a concentration of 20 mM and added to the cell culture medium at final concentrations of 0, 2.5, 5, 10, 15 and 20 μM.

Techniques: In Vitro, CCK-8 Assay, Software, Colony Assay, Staining

Journal: Journal of Cancer

Article Title: CDK1 serves as a novel therapeutic target for endometrioid endometrial cancer

doi: 10.7150/jca.51139

Figure Lengend Snippet: RO3306 inhibited HEC-1-B cell growth through induction of apoptosis and G2/M phase arrest. (A) HEC-1-B cells were treated with RO3306 at different concentrations of 0, 5 and 10 µM for 24 or 48 h. Apoptosis assay was performed with Annexin V-FITC/PI double staining using FACS analysis. (B) HEC-1-B cells were treated with RO3306 at different concentrations of 0, 5 and 10 µM for 24 or 48 h. Cell cycle assay was performed with PI staining using FACS analysis. The representative images of 3 independent experiments were shown. (C) Quantification of B. Results are the means (n = 3).

Article Snippet: CDK1 inhibitor RO3306 (Selleck Chemicals, Houston, USA) was dissolved in DMSO at a concentration of 20 mM and added to the cell culture medium at final concentrations of 0, 2.5, 5, 10, 15 and 20 μM.

Techniques: Apoptosis Assay, Double Staining, Cell Cycle Assay, Staining

Journal: Journal of Cancer

Article Title: CDK1 serves as a novel therapeutic target for endometrioid endometrial cancer

doi: 10.7150/jca.51139

Figure Lengend Snippet: RO3306 inhibited the growth of HEC-1-B cells in vivo . (A) The increment of xenograft tumor volume over time in xenograft models of HEC-1-B cells with two different treatments (vehicle or RO3306). Drugs were oral gavage administered once every 2 days and DMSO was served as vehicle control. The data shown represent mean ± SD from four samples (*p <0.05; **p <0.01; ***p <0.001; ****p <0.0001). (B) The changes of body weight of mice in two groups (the control group and the RO3306 treatment group). The data shown represent mean ± SD from four samples (p >0.05). (C) The tumor weights of the two groups (the control group and the RO3306 treatment group) at the end of the study (*p < 0.05). (D) Gross images of xenograft tumor masses of the two groups (the control group and the RO3306 treatment group) at the end of the study.

Article Snippet: CDK1 inhibitor RO3306 (Selleck Chemicals, Houston, USA) was dissolved in DMSO at a concentration of 20 mM and added to the cell culture medium at final concentrations of 0, 2.5, 5, 10, 15 and 20 μM.

Techniques: In Vivo, Control

Journal: Journal of Cancer

Article Title: CDK1 serves as a novel therapeutic target for endometrioid endometrial cancer

doi: 10.7150/jca.51139

Figure Lengend Snippet: CDK1 IHC scores in nucleus and cytoplasm of normal endometrium and endometrial cancer

Article Snippet: CDK1 inhibitor RO3306 (Selleck Chemicals, Houston, USA) was dissolved in DMSO at a concentration of 20 mM and added to the cell culture medium at final concentrations of 0, 2.5, 5, 10, 15 and 20 μM.

Techniques:

Journal: Cancer Research

Article Title: NEIL3 Prevents Senescence in Hepatocellular Carcinoma by Repairing Oxidative Lesions at Telomeres during Mitosis

doi: 10.1158/0008-5472.CAN-20-1028

Figure Lengend Snippet: NEIL3 recruits APE1 to TIFs during mitosis. A, Images of NEIL3 foci (red) and telomere (TTAGGG; green) colocalization by anti-NEIL3 IF and telo-FISH in HEP3B cells. Cells were treated with DMSO, 24-hour vincristine (VCR; 20 nmol/L), 24-hour vincristine (20 nmol/L) + 24-hour Reversine (REV; 0.5 μmol/L) or 24-hour RO-3306 (10 μmol/L). Scale bar, 5 μm. B, Quantification of NEIL3 foci–positive cells (top left), TIFs (top right), NEIL3 on telomere (bottom left), and NEIL3-positive TIFs (bottom right) in HEP3B cells. Means ± SEM; at least 200 cells were analyzed. C, Images of NEIL3 foci (red), telomere (TTAGGG, green), and γH2AX foci (cyan) colocalization by anti-NEIL3, PNA telC probe, and anti-γH2AX IF in HEP3B metaphase spread. Cells were treated with 24-hour vincristine (20 nmol/L) or synchronized in 1-hour colcemid (20 ng/mL) with 30 min KBrO3 (5 mmol/L) or DMSO. White arrowheads, NEIL3/telomere/γH2AX colocalization. Scale bar, 5 μm. D, Quantification of relative TIFs (left), NEIL3 on telomere (middle), or NEIL3-positive TIFs (right) in HEP3B metaphase spread. Means ± SEM; at least 20 metaphase cells were analyzed in each group. E, Quantification of APE1 foci on telomere (left) and APE1/NEIL3 colocalization (right) in HEP3B metaphase spread. Cells were treated with 24-hour vincristine (20 nmol/L) or synchronized in 1-hour colcemid (20 ng/mL) with 30-minute KBrO3 (5 mmol/L) or DMSO. Means ± SEM; at least 20 metaphase cells were analyzed in each group. F, Quantification of APE1 on telomere (left) and APE1-positive TIFs (right) after NEIL1, NEIL2, or NEIL3 silencing in HEP3B metaphase spread. Means ± SEM; at least 20 metaphase cells were analyzed in each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Inhibitors used are as follows: vincristine (Sigma), Colcemid (Gibco), RO-3306 (CDK1 inhibitor, MedChemExpress), Reversine (GSK923295, Axon MedChem), APE1 inhibitor (Sigma, CAS 6960–45–8), PARP inhibitor (olaparib, SelleckChem, 763113–22–0).

Techniques:

Journal: Cancer Research

Article Title: NEIL3 Prevents Senescence in Hepatocellular Carcinoma by Repairing Oxidative Lesions at Telomeres during Mitosis

doi: 10.1158/0008-5472.CAN-20-1028

Figure Lengend Snippet: NEIL3 repairs telomere damage through base excision repair in mitotic cells. A, Schematic of mitotic telomere damage repair assay. Cells were treated with 10 μmol/L RO-3306 for 16 hours to synchronize cells in G 2 phase. No damage was induced in group 0 and oxidative damage was induced in groups A–C . Group A represents no repair, group B 1 hour of repair, and group C 1 hour of repair in the presence of inhibitors. DNA repair at telomeres were analyzed by META-FISH. B, Images of APE1 foci (red), telomere PNA probe (TTAGGG, green), and γH2AX (cyan) by anti-APE1, anti-γH2AX IF, and telo-FISH in HEP3B metaphase spread. Cells were treated as indicated in A with 10 μmol/L APE1 inhibitor or 10 μmol/L PARP inhibitor (olaparib). White arrowheads, APE1/telomere/γH2AX colocalization. Scale bar, 5 μm. C, Quantification of TIFs (left) and APE1-positive TIFs (right) in HEP3B metaphase spread described in B . Means ± SEM; at least 20 metaphase cells were analyzed in each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. D, Quantification of TIFs (top) and APE1-positive TIFs (bottom) in HEP3B metaphase spread described in A . Cells were transfected with NT siRNA and siRNA targeting PolB or NEIL3 for 72 hours. Means ± SEM; at least 20 metaphase cells were analyzed in each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001. E, Telomere qPCR of NEIL1 digestion in HEP3B cells transfected with NEIL3 siRNA s2, s4, or nontargeting control (siCtrl) for 72 hours. After transfection, cells were synchronized in G 1 with 2 mmol/L thymidine, followed by synchronization in G 2 with RO-3306. Cells were released into mitosis and treated with 2.5 mmol/L KBrO 3 for 30 minutes, followed by recovery for 60 minutes. Controls are shown with no treatment (untreated) and with no recovery period (no repair). Values were normalized to untreated siCtrl and represent the mean ± SEM from three experiments. ns, nonsignificant; **, P < 0.01, one-way ANOVA analysis.

Article Snippet: Inhibitors used are as follows: vincristine (Sigma), Colcemid (Gibco), RO-3306 (CDK1 inhibitor, MedChemExpress), Reversine (GSK923295, Axon MedChem), APE1 inhibitor (Sigma, CAS 6960–45–8), PARP inhibitor (olaparib, SelleckChem, 763113–22–0).

Techniques: Transfection, Control

Journal: Nucleic Acids Research

Article Title: CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression

doi: 10.1093/nar/gku263

Figure Lengend Snippet: Suv39H1 can form complex with CDK2 but not with CDK1 in vivo. ( A ) Potential phosphorylation sites of human Suv39H1. Putative phosphorylation sites are shown in black and the phosphorylation site with the CDK1 or CDK2 consensus motif (S391 residue) is shown in red. ( B and C ) Suv39H1 interacts with CDK2 but not with CDK1 in vivo . Total cell lysates from 293T cells co-transfected with Myc-Suv39H1 and Flag-CDK1 or Flag-CDK2 (in panel (B)) or Flag-Suv39H1 and Myc-CDK1 or Myc-CDK2 (in panel (C)) were co-immunoprecipitated (IP) using anti-Myc agarose beads. The IP fractions were analyzed by western blotting using anti-Myc or anti-Flag antibody. The expression of each gene in whole cell lysate (WCL) was confirmed by western blot analysis with anti-Flag or anti-Myc antibodies. ( D ) In vitro kinase assay with purified MBP-fused Suv39H1-WT or MBP-Suv39H1-S391A mutant protein. Purified MBP-Suv39H1-WT and MBP-Suv39H1-S391A mutant proteins were incubated with gamma- 32 P-ATP and CDK2/cyclin complexes. The phosphorylation was visualized by exposure to X-ray film. The data showed that MBP-Suv39H1-WT was phosphorylated by both CDK2/cyclin A2 and CDK2/cyclin E1 complexes but not MBP-Suv39H1-S391A mutant.

Article Snippet: The CDK1 inhibitor IVRO-3306 (217699) and CDK2 inhibitor II (219445) were purchased from Merck Millipore (Darmstadt, Germany), dissolved in dimethyl sulfoxide (DMSO).

Techniques: In Vivo, Phospho-proteomics, Residue, Transfection, Immunoprecipitation, Western Blot, Expressing, In Vitro, Kinase Assay, Purification, Mutagenesis, Incubation

Journal: Nucleic Acids Research

Article Title: CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression

doi: 10.1093/nar/gku263

Figure Lengend Snippet: CDK2 phosphorylates Suv39H1 at Ser-391 in vivo. ( A ) Suv39H1 phosphorylation at Ser-391 was decreased by treatment of CDK2 inhibitor but not by CDK1 inhibitor. The 293T cells stably expressing Flag-Suv39H1 were treated with CDK1 or CDK2 inhibitor for 20 h and 12 h, respectively. Total cell lysates were immunoprecipitated using anti-Flag M2 and analyzed by western blotting using ant anti-phosphoSuv39H1 (pS391). ( B ) The shRNA-based CDK2 knockdown resulted in decrease of Suv39H1 phosphorylation at Ser-391 residue whereas the phosphorylation was little changed by CDK1 knockdown. The 293T cells stably expressing Flag-empty vector (negative control) or Flag-Suv39H1 were infected with lentivirus expressing CDK1 shRNA or CDK2 shRNA. Cell lysates were immunoprecipitated using anti-Flag M2 and then analyzed by western blotting using ant anti-phosphoSuv39H1 (pS391). ( C ) Phosphorylation of Suv39H1 at Ser-391 was decreased by overexpression of a dominant negative mutant of CDK2 but not CDK1. The 293T cells were co-transfected with Flag-Suv39H1-WT and Myc-CDK2 or its dominant negative mutant Myc-CDK2 D145N, or with Myc-CDK1 or its dominant mutant derivatives (CDK1 K33R or CDK1 D146N). Total lysates were immunoprecipitated using anti-Flag M2 agarose beads and analyzed by western blotting using anti-pSer-391 antibody. ( D ) Ser-391 of Suv39H1 is phosphorylated predominantly during S phase and maintained during S-G2-M phases. To profile the cell-cycle-dependent phosphorylation of Suv39H1, the HeLa cell lines stably expressing Flag-tagged Suv39H1-WT were treated by nocodazole and release. Total cell lysates of cells released from cell-cycle blockage were subjected to immunoprecipitation using anti-Flag M2 agarose beads and analyzed by western blotting using anti-pSer-391 antibody.

Article Snippet: The CDK1 inhibitor IVRO-3306 (217699) and CDK2 inhibitor II (219445) were purchased from Merck Millipore (Darmstadt, Germany), dissolved in dimethyl sulfoxide (DMSO).

Techniques: In Vivo, Phospho-proteomics, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, shRNA, Knockdown, Residue, Plasmid Preparation, Negative Control, Infection, Over Expression, Dominant Negative Mutation, Transfection, Mutagenesis

Journal: Journal of bioinformatics and systems biology : Open access

Article Title: CDKs Functional Analysis in Low Proliferating Early-Stage Pancreatic Ductal Adenocarcinoma.

doi: 10.26502/jbsb.5107060

Figure Lengend Snippet: Figure 2: A. Log2 mRNA expression level of MKI67 in the high group (≥ 75th percentile) and low group (<75th percentile) in early-stage PDAC patients. B. Patients in the high MKI67 group (MKI67 high) had shorter median survival compared to the low MKI67 group (MKI67 low). Pearson's correlation analysis was performed to analyze the mRNA expression correlation between MKI67 and CDK1 (C), CDK2 (D), CDK4 (E), and CDK6 (F).

Article Snippet: PANC1 and MiaPaca II cells were treated with CDK1 inhibitors (10 uM CDK1-IN-2, MedChemExpress HY-112463; or 5 uM RO-3306, MedChemExpress HY12529) or vehicle for 24h or 48hrs in the presence/absence of 10%FBS.

Techniques: Expressing

Journal: Nature Communications

Article Title: Plk1 relieves centriole block to reduplication by promoting daughter centriole maturation

doi: 10.1038/ncomms9077

Figure Lengend Snippet: ( a , b ) Centrioles of C1–GFP-expressing cells arrested in G2 by Cdk1 inhibitor RO-3306 (RO) were first analysed by 5 min time lapse to determine the pattern of centriole movement (expressed as the average distance between the mother and daughter C1–GFP signals). The same centrioles were then fixed and analysed by electron microscopy. Mother–daughter centriole pairs with average C1–GFP distance ≤700 nm were found in orthogonal orientation and at the wall-to-wall distance <80 nm. Daughter centrioles ≥80 nm from the wall of the mother centrioles were found disoriented. Scale bars, 500 nm. ( c , d ) Daughter centrioles in the centrosomes with C1–GFP distances ≤700 nm are associated with a substantial amount of PCM (as evidenced by a gamma tubulin signal) and with licensing factor Cep152. Accumulation of Cep152 precedes the degradation of Sas6, separation of two PCMs and loss of orthogonal orientation between the centrioles. Scale bar, 1 μm. ( e ) Quantification of daughter centriole-associated gamma tubulin and Cep152. Only centrioles with mother centriole perpendicular to the coverslip were measured. For each centrosome, two signal lengths were measured from the centre of a gamma tubulin or Cep152 toroid organized by the mother centriole, until the end of the signal associated with the daughter centriole (determined as a signal half maximum). Centrioles in cells arrested in G2 and treated with Plk1 inhibitor BI2536 (RO+BI) were measured as a control.

Article Snippet: These cells were then either cultured without any further treatment and analysed at appropriate time points, or treated with 2 mM HU (Sigma) to induce S-phase arrest, or with 9 μM Cdk1 inhibitor RO-3306 (Tocris Biosciences) to induce G2 arrest .

Techniques: Expressing, Electron Microscopy, Control