Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: Specific inhibition of CDK7 by 1-NMPP1 prevents DNA synthesis (A), pRb phosphorylation (B), CDK4/6 pRb-kinase activity (C) and CDK4 activating phosphorylation (D). HCT116 WT or K7AS (K7as/as) cells were stimulated (+) or not stimulated (−) with FBS for the indicated times in the absence (−) or presence (+) of 1-NMPP1. (A) DNA synthesis was evaluated from duplicate dishes by counting the percentage of cells having incorporated BrdU during the last 30 min of stimulation. (B) Western blotting analysis with the indicated antibodies from whole-cell lysates. (C) Cell lysates were immunoprecipitated (IP) with anti-cyclin D1 (D1), anti-cyclin D3 (D3) or anti-p21 (p21) antibodies and were assayed for their pRb-kinase activity, separated by SDS-PAGE and immunoblotted with the indicated antibodies (C). (D) The same immunoprecipitates were separated by 2D gel electrophoresis followed by immunodetection using antibodies directed against CDK4 or T172-phosphorylated CDK4. Arrows, T172-phosphorylated form of CDK4.

Article Snippet: The beads were resuspended in 50 µl of CAK buffer containing protease and phosphatase inhibitors and 2 mM ATP and incubated at 30°C for 1 h with or without 1 µg of recombinant kinases: CDK2-cyclin E1, CDK2-cyclin A2 and CDK4-cyclin D3 (Carna Biosciences); cyclin H-CDK7-MAT1 (Upstate); CDK4-cyclin D1 and CDK6-cyclin D1 (ProQinase) .

Techniques: Inhibition, DNA Synthesis, Activity Assay, Western Blot, Immunoprecipitation, SDS Page, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: Effect of acute inhibition of CDK7 by 1-NMPP1 on CDK4 activity (A) and phosphorylation (B). (A,B) HCT116 K7AS cells were stimulated with FBS for 5 h and 1-NMPP1 was then added for 1 or 3 h. Cell lysates were immunoprecipitated (IP) with anti-cyclin D1 (D1), or anti-p21 antibodies, assayed for their pRb-kinase activity, separated by SDS-PAGE and immunoblotted with the indicated antibodies (A), or separated by 2D gel electrophoresis followed by CDK4 immunodetection (B). In (B), T160 phosphorylation of CDK2 immunodetected from whole cell extracts is shown for comparison.

Article Snippet: The beads were resuspended in 50 µl of CAK buffer containing protease and phosphatase inhibitors and 2 mM ATP and incubated at 30°C for 1 h with or without 1 µg of recombinant kinases: CDK2-cyclin E1, CDK2-cyclin A2 and CDK4-cyclin D3 (Carna Biosciences); cyclin H-CDK7-MAT1 (Upstate); CDK4-cyclin D1 and CDK6-cyclin D1 (ProQinase) .

Techniques: Inhibition, Activity Assay, Immunoprecipitation, SDS Page, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection, Comparison

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: (A) HCT116 K7AS cells were stimulated (+) or not stimulated (−) with fetal bovine serum (FBS) for the indicated times in the absence (−) or presence (+) of 1-NMPP1. Cell lysates were immunoprecipitated (IP) with anti-p21, anti-cyclin D1 (D1) or anti-cyclin D3 (D3) antibodies and separated by 2D gel electrophoresis followed by p21 immunodetection. (B) CHO cells were transfected with a p21-expressing plasmid alone (left) or with plasmids encoding cyclin D3, CDK4-HA and p21 (right). Cell lysates were immunoprecipitated (IP) with anti-p21 (p21) or anti-cyclin D3 (D3) antibodies and incubated with the indicated recombinant kinases and ATP, before separation by 2D gel electrophoresis and p21 immunodetection. Arrows indicate the main phosphorylated forms of p21 as identified in . (C) Schematic representation and comparison of p21 patterns from HCT116 K7AS cells that were restimulated for 3 and 20 h ( in vivo ) and from cyclin D3-CDK4-p21 complexes produced in CHO cells and phosphorylated by recombinant CDK2-cyclin E and CDK2-cyclin A ( in vitro ). The observed p21 forms are labeled according to their number of phosphorylations and the identified phosphorylated residues (e.g., 2P 98,130 means p21 phosphorylated at both S98 and S130; X, Y, unidentified phosphorylated sites).

Article Snippet: The beads were resuspended in 50 µl of CAK buffer containing protease and phosphatase inhibitors and 2 mM ATP and incubated at 30°C for 1 h with or without 1 µg of recombinant kinases: CDK2-cyclin E1, CDK2-cyclin A2 and CDK4-cyclin D3 (Carna Biosciences); cyclin H-CDK7-MAT1 (Upstate); CDK4-cyclin D1 and CDK6-cyclin D1 (ProQinase) .

Techniques: Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection, Transfection, Expressing, Plasmid Preparation, Incubation, Recombinant, Comparison, In Vivo, Produced, In Vitro, Labeling

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: (A) p21 stabilizes cyclin D3-CDK4 complexes and inhibits their activity depending on its expression level. CHO cells were transfected with plasmids encoding cyclin D3 and CDK4-HA with or without p21 at high (p21) or low (p21 1/10) expression level. Cell lysates (WCE) were immunoprecipitated (IP) with anti-cyclin D3 (D3) or anti-p21 antibodies, assayed for their pRb-kinase activity, separated by SDS-PAGE and immunoblotted with the indicated antibodies. (B) p21 expressed alone is essentially unphosphorylated. CHO cells were transfected with a plasmid encoding p21 at high (p21) or low (p21 1/10) expression level. Cell lysates were immunoprecipitated (IP) with anti-p21 antibody and separated by 2D gel electrophoresis followed by p21 immunodetection. (C) Co-expression of cyclin D3-CDK4 induces the phosphorylation of p21, largely independently of CDK4 activity, whereas stronger expression of p21 inhibits phosphorylations of both p21 and CDK4. CHO cells were transfected with plasmids encoding cyclin D3 and CDK4-HA or its inactive mutants (T172A, K35R) with or without p21 at high (p21) or low (p21 1/10) expression level. Cell lysates were immunoprecipitated (IP) with anti-cyclin D3 antibody and separated by 2D gel electrophoresis followed by CDK4 and p21 immunodetection. (D) Identification of p21 phosphorylation sites. CHO cells were transfected with plasmids encoding cyclin D3, CDK4-HA and wild type (WT) or mutated (S130A, S98A) p21 at low (p21 1/10) expression level. Cell lysates were immunoprecipitated (IP) with anti-cyclin D3 antibody and separated by 2D gel electrophoresis followed by p21 immunodetection. Black arrows, T172-phosphorylated CDK4. Colored arrows indicate phosphorylated forms of p21.

Article Snippet: The beads were resuspended in 50 µl of CAK buffer containing protease and phosphatase inhibitors and 2 mM ATP and incubated at 30°C for 1 h with or without 1 µg of recombinant kinases: CDK2-cyclin E1, CDK2-cyclin A2 and CDK4-cyclin D3 (Carna Biosciences); cyclin H-CDK7-MAT1 (Upstate); CDK4-cyclin D1 and CDK6-cyclin D1 (ProQinase) .

Techniques: Activity Assay, Expressing, Transfection, Immunoprecipitation, SDS Page, Plasmid Preparation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: HCT116 K7AS cells were stimulated (+) or not stimulated (−) with fetal bovine serum (FBS) for the indicated times in the absence (−) or presence (+) of roscovitine (rosco) (A,B) or CR8 (C). (A) Western blotting analysis with the indicated antibodies from whole-cell lysates. (B,C) Cell lysates were immunoprecipitated (IP) with anti-cyclin D1 (D1), anti-cyclin D3 (D3) or anti-p21 antibodies and separated by 2D gel electrophoresis followed by immunodetection of CDK4 and p21. Black arrows, T172-phosphorylated CDK4; green arrows, S130-phosphorylated p21.

Article Snippet: The beads were resuspended in 50 µl of CAK buffer containing protease and phosphatase inhibitors and 2 mM ATP and incubated at 30°C for 1 h with or without 1 µg of recombinant kinases: CDK2-cyclin E1, CDK2-cyclin A2 and CDK4-cyclin D3 (Carna Biosciences); cyclin H-CDK7-MAT1 (Upstate); CDK4-cyclin D1 and CDK6-cyclin D1 (ProQinase) .

Techniques: Western Blot, Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: (A,B) HCT116 K7AS cells were stimulated (+) or not stimulated (−) with fetal bovine serum (FBS) for 5 h in the absence (−) or presence (+) of the CDK4/6 inhibitor PD0332991. Cell lysates were immunoprecipitated (IP) with anti-cyclin D1 (D1) or anti-p21 antibodies, assayed for their pRb-kinase activity, separated by SDS-PAGE and immunoblotted with the indicated antibodies (A), or separated by 2D gel electrophoresis followed by CDK4 and p21 immunodetection (B). (C) CHO cells were transfected with plasmids encoding cyclin D3, CDK4-HA and p21 at low (p21 1/10) expression level (see ), in the absence (DMSO as vehicle) or presence of CDK inhibitors: roscovitine (rosco), PD0332991 (1 µM) or their combination to inhibit both CDK2 and CDK4. Cell lysates were immunoprecipitated (IP) with anti-cyclin D3 (D3) antibody and separated by 2D gel electrophoresis followed by CDK4 and p21 immunodetection. Black arrows, T172-phosphorylated CDK4; colored arrows, phosphorylated forms of p21.

Article Snippet: The beads were resuspended in 50 µl of CAK buffer containing protease and phosphatase inhibitors and 2 mM ATP and incubated at 30°C for 1 h with or without 1 µg of recombinant kinases: CDK2-cyclin E1, CDK2-cyclin A2 and CDK4-cyclin D3 (Carna Biosciences); cyclin H-CDK7-MAT1 (Upstate); CDK4-cyclin D1 and CDK6-cyclin D1 (ProQinase) .

Techniques: Immunoprecipitation, Activity Assay, SDS Page, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection, Transfection, Expressing

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: (A) HCT116 K7AS cells were transfected with plasmids encoding cyclin D3 and CDK4-HA in the absence or continuous presence of 1-NMPP1 for 48 h. Cell lysates were immunoprecipitated (IP) with anti-cyclin D3 or anti-p21 antibodies and separated by 2D gel electrophoresis followed by immunoblotting with anti-CDK4 antibody, which detected both ectopic and endogenous CDK4. (B) HCT116 K7AS cells were infected during 24 h by lentiviruses encoding Tet-On inducible wt or S178P CDK6-Flag with or without lentiviruses encoding Tet-On inducible cyclin D3. The expression of these proteins in the continuous presence (+) or absence (−) of 1-NMPP1 was induced by doxycycline 16 h prior to cell restimulation by fetal bovine serum (FBS) for 16 h. Western blotting detection of endogenous and ectopic (arrow) CDK6 from whole cell extracts is shown in the upper panel (NI, not infected). Cell lysates were immunoprecipitated (IP) with anti-Flag, anti-cyclin D3 or anti-p21 antibodies and separated by 2D gel electrophoresis followed by immunoblotting with anti-CDK6 antibody, which detected both ectopic and endogenous (e) CDK6. (C) T172 phosphorylation of endogenous CDK4 resists inhibition of CDK2 and CDK7 by roscovitine in p21-null (p21−/−) cells. Roscovitine (rosco; +) or vehicle (−) were administered for 16 h to asynchronously growing wild-type (WT) and p21−/− HCT116 cells cultured in parallel. Cell lysates were immunoprecipitated (IP) with anti-cyclin D1 (D1) or anti-cyclin D3 (D3) antibodies and separated by 2D gel electrophoresis followed by CDK4 immunodetection. Arrows, phosphorylated forms of CDK4 and CDK6-Flag; arrowheads in (B), unphosphorylated form of CDK6-Flag.

Article Snippet: The beads were resuspended in 50 µl of CAK buffer containing protease and phosphatase inhibitors and 2 mM ATP and incubated at 30°C for 1 h with or without 1 µg of recombinant kinases: CDK2-cyclin E1, CDK2-cyclin A2 and CDK4-cyclin D3 (Carna Biosciences); cyclin H-CDK7-MAT1 (Upstate); CDK4-cyclin D1 and CDK6-cyclin D1 (ProQinase) .

Techniques: Transfection, Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Infection, Expressing, Inhibition, Cell Culture, Immunodetection

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: CDK7 inhibition reveals novel positive feedbacks mediated by S130 phosphorylation of p21, which is catalyzed by active CDK4 and CDK2 to permit T172 phosphorylation and activation of CDK4 complexes stabilized by p21 binding. S130 phosphorylation of p21 thus amplifies CDK4 activation and subsequently leads to increased degradation of p21, which in turn facilitates CDK2 activation. Those retrocontrols of CDK4 activation should contribute to explaining the irreversibility of the cell cycle commitment at the R point. Green/red colors indicate a final positive/negative influence on R point passage. 1 , 2 , two different levels of CDK7 action demonstrated by the present observations. CDK4 AK?, hypothetical CDK4-activating kinase(s) phosphorylating p21-unbound CDK4 and S178P CDK6 during CDK7 inhibition.

Article Snippet: The beads were resuspended in 50 µl of CAK buffer containing protease and phosphatase inhibitors and 2 mM ATP and incubated at 30°C for 1 h with or without 1 µg of recombinant kinases: CDK2-cyclin E1, CDK2-cyclin A2 and CDK4-cyclin D3 (Carna Biosciences); cyclin H-CDK7-MAT1 (Upstate); CDK4-cyclin D1 and CDK6-cyclin D1 (ProQinase) .

Techniques: Inhibition, Activation Assay, Binding Assay

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 1. miR-29c regulates the expression of cyclin E at the posttranscriptional level by targeting 3# UTR of cyclin E mRNA in ESCC cells. (A) The public miRNA database (microRNA Targets Version 5) predicted that cyclin E might be a target for miR-29c, and the 3# UTR of human cyclin E mRNA contains a highly conserved binding site from Position 470 to 492 for miR-29c. (B) The full-length 3# UTR of cyclin E complementary DNA containing miR-29c-binding site was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Wt plasmid (Wt). The full-length 3# UTR of cyclin E complementary DNA (cDNA) deleted 22 nt miR-29c-binding sequence was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Mut plasmid (Mut). (C) KYSE150 cells and EC9706 cells were transfected with 800 ng Wt or Mut reporter plasmid and the increasing doses of Pre-miR-29c or Pre-Scramble (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). After transfected for 24 h, luciferase activity was measured by a dual-luciferase reporter assay. The result was expressed as relative luciferase activity (firefly LUC/renilla LUC). Columns, mean for three experiments; bars, SE. (D) EC9706 cells and KYSE150 cells were transfected with either 30 nmol/l Pre-miR-29c or Pre-Scramble for 48 h. Cyclin E protein levels were measured by western blotting and cyclin E mRNA levels were measured by reverse transcription–PCR.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Reporter Assay, Western Blot, Reverse Transcription

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 2. Inverse correlation between miR-29c expression and cyclin E protein in ESCC cell lines. (A) miR-29c expression in KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells was analyzed by quantitative real-time PCR. The results were presented as relative miR-29c expression, RNU6B served as internal control. The relative value of miR-29c expression of KYSE450 is set at 1. (B and C) The cell lysates of KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells were prepared and analyzed by western blotting. The density of each protein band was quantified using LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. The relative value of cyclin E expression in KYSE450 is set at 1. (D) KYSE150 cells were transfected with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). Forty-eight hours after transfection, miR-29c level was detected by using quantitative real-time PCR. (E and F) The expression of cyclin E was measured by western blotting, after transfecting KYSE150 cells with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l) for 48 h. The density of each protein band was quantified by LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Software, Transfection

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 3. miR-29c induced G1/S cell cycle arrest by suppression of cyclin E expression. (A) EC9706 and KYSE150 cells were transfected with 30 nmol/ l Pre-miR-29c, Pre-Scramble or only Lipofectmine 2000 (Mock). Forty-eight hours after transfection was treated with 100 ng/ml nocodazole for 20 h, cells were collected for cell cycle analysis by propidium iodide staining and flow cytometer analysis. The percentage value of G1 fraction between Pre-miR-29c transfected cells and Pre-Scramble or Mock transfected cells was analyzed. P , 0.01. (B) EC9706 cells were transfected with 30 nmol/l Pre-miR-29c along with the expression plasmid pEF-cyclin E, which contains cyclin E open reading frame without 3# UTR. Forty-eight hours after transfection, cells were treated with 100 ng/ml nocodazole for 20 h. The percentage of cells in G1/G0 was determined by flow cytometer. (C) EC9706 cells and KYSE150 cells were transfected with 30 nmol/l Pre-miR-29c, Pre-Scramble or Mock for 48 h. The cells were collected for western blotting using antibody against cyclin D1, cyclin D2, CDK2 and CDK6. b-Actin was used as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Transfection, Cell Cycle Assay, Staining, Cytometry, Plasmid Preparation, Western Blot, Control

Journal: Physiological Research

Article Title: miR-491-5p Inhibits Emilin 1 to Promote Fibroblasts Proliferation and Fibrosis in Gluteal Muscle Contracture via TGF-β1/Smad2 Pathway

doi: 10.33549/physiolres.934804

Figure Lengend Snippet: Effects of miR-491-5p/Emilin 1 on proliferation of CB fibroblasts. (A) Cells were transfected with miR-491-5p mimics or mimics control and inhibitors or inhibitors control. CCK8 assay was carried out to evaluate cell viability in each group. (B) Cells were transfected with miR-491-5p mimics or mimics control, and Emilin 1 overexpression plasmid or vector control. CCK8 assay was carried out to evaluate cell viability in each group. (C) Immunoblot assay was applied to measure protein expression of Cyclin D1 and CDK2. (D) CDK4 and Cyclin D1 expression levels were determined with grayscale. All data analysis above used One-Way ANOVA analysis of variance. Data were shown as the mean ± SD, n = 3. *p<0.05 and **p<0.01.

Article Snippet: After blocking with 5 % no-fat milk that diluted in 0.1 % TBST for 1.5 h at room temperature, bands were incubated with primary antibodies against Emilin 1 (1:1000, ab243324, Abcam), CDK2 (1:1000, 18048S, Cell Signalling Technology), Cyclin D1 (1:1000, 55506S, Cell Signalling Technology), p-Smad2 (1:1000, 18338S, Cell Signalling Technology), Smad2 (1:1000, 5339S, Cell Signalling Technology), and TGF-β1 (1:1000, 3709S, Cell Signalling Technology) at 4 °C overnight.

Techniques: Transfection, Control, CCK-8 Assay, Over Expression, Plasmid Preparation, Western Blot, Expressing

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Examples of pyran-containing compounds with antibacterial , antioxidant and anticancer activities via inhibition of CDK2 [ , , ].

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Inhibition

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Redocked (yellow) and co-crystallized (baby blue) ligand ( DTQ ) in the ATP binding pocket of CDK2 after self-docking calculations.

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Binding free energy of 4d , 4k , 4f , BMS-265246 and DTQ at the active site of CDK2.

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Three-dimensional binding interactions of DTQ within the ATP binding pocket of CDK2. Hydrogen bonds (yellow dotted lines), hydrogen atoms (white), nitrogen atoms (blue), and oxygen atoms (red).

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Two-dimensional binding interactions of DTQ within the ATP binding pocket of CDK2.

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Three-dimensional binding interactions of BMS-265246 within the ATP binding pocket of CDK2. Hydrogen bond (purple dotted line), hydrogen atoms (white), nitrogen atoms (blue), fluorine atoms (light green), and oxygen atoms (red).

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Two-dimensional interactions of BMS-265246 within the ATP binding pocket of CDK2 kinase.

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Three-dimensional model of binding interactions of compound 4d after docking calculations in the ATP binding pocket of CDK2. Hydrogen bond (black lines), hydrogen atoms (white), nitrogen atoms (blue), and oxygen atoms (red).

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Two-dimensional model of binding interactions of compound 4d after docking calculations in the ATP binding pocket of CDK2.

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Three-dimensional model of binding interactions of compound 4K after docking calculations in the ATP binding pocket of CDK2. Hydrogen bond (black lines), hydrogen atoms (white), nitrogen atoms (blue), and oxygen atoms (red).

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Two-dimensional model of binding interactions of compound 4K after docking calculations in the ATP binding pocket of CDK2.

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Three-dimensional model of binding interactions of compound 4f after docking calculations in the ATP binding pocket of CDK2. Hydrogen atoms (white), nitrogen atoms (blue), chlorine atom (green), and oxygen atoms (red).

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: Two-dimensional model of binding interactions of compound 4f after docking calculations in the ATP binding pocket of CDK2.

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: In vitro evaluation of the CDK2 inhibitory efficiency of pyrans 4d and 4K as compared to the reference inhibitor BMS-265246 over a concentration range of 0.01−10 µM. Results were obtained from three independent experiments.

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: In Vitro, Concentration Assay

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: IC 50 values of 4d , 4K and BMS-265246 against CDK2.

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques:

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: In vitro quantitative determination of the concentrations of CDK2 (ng/mL) in HCT116 cells treated with pyrans 4d and 4K compared with the positive control BMS-265246 and the negative control samples.

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: In Vitro, Positive Control, Negative Control

Journal: Pharmaceuticals

Article Title: Synthesis and Evaluation of Some New 4 H -Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations

doi: 10.3390/ph15070891

Figure Lengend Snippet: The expression profiles of the CDK2 gene in the lysate of HCT-116 cancer cells treated with compounds 4d and 4 k .

Article Snippet: CDK-2 inhibition activity of the studied compounds 4d and 4k was evaluated using a commercial CDK2 ELISA Kit (cat. no.: 79599; BPS Biosciences, San Diego, CA, USA) following the manufacturer’s instructions.

Techniques: Expressing