Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Combined CDK4/6 mTOR inhibition is synergistic against glioblastoma via multiple mechanisms

doi: 10.1158/1078-0432.CCR-17-0803

Figure Lengend Snippet: (A) Enrichment analysis showing increased sensitivity of cancer lines with mutations in RB1 to an mTOR inhibitor, sirolimus. (B) Synergy scores of the combination of CDK4/6 and mTOR inhibition in two GIC lines calculated with both the Bliss and the Chou-Talalay methods. (C) 10 and 100 GICs were cultured in 24-well plates over two weeks to compare sphere formation upon treatment with vehicle, palbociclib (1 μM), everolimus (4 μM), and the combination of palbociclib and everolimus (*P < 0.05; **P < 0.001; ***P < 0.0001; one-way analysis of variance (ANOVA) with post-hoc Tukey analysis). (D) The combination of a different mTOR inhibitor temsirolimus (4 μM) and a different CDK4/6 inhibitor ribociclib (4 μM) is also synergistic against GICs (***P < 0.0001; one-way ANOVA with post-hoc Tukey analysis). (E) The combinations of palbociclib (4 μM) with mTOR siRNA and everolimus (5 μM) with CDK4/6 siRNA are also synergistic against GICs (each treatment line received either DMSO or the indicated drug and either control siRNA or a specific siRNA) (**P < 0.001; ***P < 0.0001; one-way ANOVA with post-hoc Tukey analysis). (F) Constitutively-active CDK4 and mTOR plasmids partially rescued from the effects of the combination treatment (each treatment line received either a control plasmid or the indicated active plasmid and either DMSO or the combination treatment) (*P < 0.05; two-tailed t-test). (G and H) Combined CDK4/6 and mTOR inhibition induces significant apoptosis. Shown is an immunoblot using antibodies specific for Bcl-2 and PARP. Caspase-3/7 level is increased with the three days of combined treatment. (*P < 0.05; ***P < 0.0001; one-way ANOVA with post-hoc Tukey analysis).

Article Snippet: Plasmid and siRNA transfection Constitutively-active CDK4 (RC401060, Origene) and mTOR (69010, Addgene) as well as the respective control plasmids were used for rescue experiments.

Techniques: Inhibition, Cell Culture, Control, Plasmid Preparation, Two Tailed Test, Western Blot

Journal: Cancer Research

Article Title: Cyclin-Dependent Kinase–Mediated Phosphorylation Plays a Critical Role in the Oncogenic Functions of PELP1

doi: 10.1158/0008-5472.can-10-0628

Figure Lengend Snippet: Figure 1. PELP1 is a novel substrate of interphase CDKs. A, expression status of PELP1 in the cell cycle was analyzed in IMR-90 cells (left) and NIH3T3 cells (right). Cells synchronized at G0-G1 phase were released into the cell cycle, and lysates from different time intervals were used in Western blot analysis with the 220B2 PELP1 antibody. B, total lysates from MCF7 cells grown in 10% serum were subjected to immunoprecipitation (IP) using the PELP1 antibody (left), and the CDK4 interaction was verified by Western blotting. Middle, T7-tagged PELP1-overexpressing MCF7 cells were treated with 10−8 mol/L estrogen for various periods of time, PELP1 was immunoprecipitated, and the CDK4 interaction was verified by Western blotting. Right, in vitro kinase assays for the CDK4/cyclin D complex using baculovirus-expressed, GST-tagged, full-length PELP1 as a substrate, and phosphorylation was measured by the amount of 32P incorporation. C, left, total lysates from MCF7 cells grown in 10% serum were subjected to immunoprecipitation using the PELP1 antibody and the CDK2 interaction was verified by Western blot analysis. Middle, MCF7 cells were treated with E2 for various periods of time, and the PELP1 interaction with CDK2 was analyzed by using immunoprecipitation. Right, in vitro kinase assays using CDK2/cyclin E (CyE) complex and CDK2/ cyclin A2 (CyA) complex using full-length PELP1 as a substrate.

Article Snippet: The plaslutathione S-transferase (GST)–PELP1 deletions (16), uc (17), and GFP-PELP1 (16) were described previously. sion vectors for p16INK4A, CDK4, CDK2, and cyclin E urchased from Addgene, Inc.

Techniques: Expressing, Western Blot, Immunoprecipitation, In Vitro, Phospho-proteomics

Journal: Cancer Research

Article Title: Cyclin-Dependent Kinase–Mediated Phosphorylation Plays a Critical Role in the Oncogenic Functions of PELP1

doi: 10.1158/0008-5472.can-10-0628

Figure Lengend Snippet: Figure 2. Identification of phosphorylation sites and generation of phospho-specific antibody. A, bacterially expressed GST-PELP1 deletions were used as substrates for an in vitro kinase assay using CDK4/cyclin D1 (left), CDK2/cyclin E (middle), and CDK2/cyclin A2 (right), and PELP1 domains phosphorylated by each kinase complex were identified by using autoradiography. B, identification of CDK phosphorylation sites using site-directed mutagenesis (serine to alanine). Various single MTs in the region of interest were used along with respective PELP1-WT deletion fragments. Loss of 32P incorporation revealed the successful identification of phosphorylation sites. C, Western blot analysis of native, WT-tagged PELP1 and tagged phospho-PELP1-MT with the Ser991 phospho-PELP1 antibody in the presence or absence of the phosphopeptide. D, MCF7 cells were either arrested and released by E2 stimulation (left) or synchronized into the G1-S boundary by double-thymidine block and released into the cell cycle by addition of thymidine-free medium (right), and phosphorylation status of PELP1 was analyzed by using the phospho-Ser991 antibody.

Article Snippet: The plaslutathione S-transferase (GST)–PELP1 deletions (16), uc (17), and GFP-PELP1 (16) were described previously. sion vectors for p16INK4A, CDK4, CDK2, and cyclin E urchased from Addgene, Inc.

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Autoradiography, Mutagenesis, Western Blot, Blocking Assay

Journal: Oncology letters

Article Title: P16 INK4A is required for cisplatin resistance in cervical carcinoma SiHa cells.

doi: 10.3892/ol.2014.2814

Figure Lengend Snippet: Figure 2. P16INK4A‑CDK4 interaction is upregulated in SiHa‑DDP cells with pRb inactivation. (A) Western blot analysis revealed the changes in P16, cyclin D1 and pRb protein expression, indicating an inverse cor relation with P16 and cyclin D1, pRb in DDP‑resistant SiHa‑DPP cells. (B) Co‑immunoprecipitation was conducted to evaluate the enhanced interaction between P16 and CDK4 in SiHa‑DDP cells when compared with SiHa cells. DDP, cisplatin; Rb, retinoblastoma protein; pRb, phosphorylated retinoblastoma protein; CDK, cyclin‑dependent kinase.

Article Snippet: The membranes were then incubated with human anti-mouse monoclonal p16 antibody (Sigma-Aldrich, St. Louis, MO, USA), human anti-mouse polyclonal pRb and β-actin antibodies (Cell Signaling Technology Inc., Danvers, MA, USA), human anti-rabbit monoclonal CDK4 antibody (Cell Signaling Technology, Inc.) overnight at 4 ̊C.

Techniques: Western Blot, Expressing

Journal: Journal of Biomedical Research

Article Title: Activation of p38/HSP27 pathway counters melatonin-induced inhibitory effect on proliferation of human gastric cancer cells

doi: 10.7555/JBR.33.20180066

Figure Lengend Snippet: A: Cell proliferation was evaluated by MTT assays after melatonin treatment for the indicated doses and time. Values were determined by three independent experiments. * italic P /italic 0.05, ** italic P /italic 0.01. B: BGC-823 cells were stimulated with 1 mmol/L melatonin for 48 hours, and cell cycle was measured by flow cytometry analysis. C: The expression of cyclin D1 and CDK4 were determined by immunobloting analysis. -Actin was used as a loading control. * italic P /italic 0.05, ** italic P /italic 0.01. D: Melatonin-induced apoptosis was measured by flow cytometry analysis.

Article Snippet: The following antibodies were used: rabbit anti-p38 antibody, rabbit anti-p-p38 antibody, mouse anti-HSP27 antibody, rabbit anti-p-HSP27 antibody, rabbit anti-CDK4 antibody and rabbit anti-cyclin D1 antibody (Cell Signaling, Danvers, MA, USA), rabbit anti-GAPDH antibody (Santa Cruz, Santa Cruz, CA, USA), mouse anti-β-actin antibody (Sigma, USA).

Techniques: Flow Cytometry, Expressing, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Activation of the CKI-CDK-Rb-E2F Pathway in Full Genome Hepatitis C Virus-expressing Cells

doi: 10.1074/jbc.m312822200

Figure Lengend Snippet: FIG. 5. Activation of E2F and CDK occurs in RzM6 cells after 44 days of passage. A, DHFR promoter activity in RzM6, Fse-Hep, and Age-Hep cells. Each reaction was standardized using the Renilla luciferase reporter plasmid (relative luciferase activity). The ratio of the -fold increase was calculated by the division of each relative luciferase activity in cells prior to HCV expression. -Fold activation of relative luciferase activity is given as a ratio. The results represent the average of 3 wells in two independent experiments. B, CDK2 and CDK4/6 activities in Rz2-18 and RzM6 cells on days 0, 8, and 44. NRS, immunoglobulin derived from normal rabbit serum; NMS, immunoglobulin derived from normal mouse serum. The percentage of each kinase activity relative to day 0 as quantitated using a phospho-imager plate is shown. tr.Rb, truncated Rb. C, stability of the p21WAF1/CIP1 and Rb proteins characterized by Western blotting after cycloheximide treatment for the indicated times.

Article Snippet: Antibodies against p53 (Novo Castra); p21WAF1/CIP1 and EB1 (Transduction Laboratories); RhoA, interferon (IFN) regulatory factor-1, Rho guanine nucleotide dissociation inhibitor, p27KIP1, p16INK, cyclin A, cyclin D1, cyclin E, CDK2, and CDK4 (Santa Cruz Biotechnology); phospho-CDC2 (New England Biolabs Inc.); actin (Roche Applied Science); and Rb (Pharmingen) were purchased and utilized according to the manufacturers’ protocols.

Techniques: Activation Assay, Activity Assay, Luciferase, Plasmid Preparation, Expressing, Derivative Assay, Western Blot

Journal: Journal of pharmaceutical and biomedical analysis

Article Title: Integrated metabolomics and proteomics analyses to reveal anticancer mechanism of hemp oil extract in colorectal cancer.

doi: 10.1016/j.jpba.2024.116379

Figure Lengend Snippet: Fig. 5. HOE arrests the cell cycle at the G1 phase. A: Flow cytometric analysis showed that HOE arrested the SW620 cells at the G1 phase. B: The statistical analysis result of the cell flow cytometric analysis. C: Results of western blotting showed that HOE decreased the protein expression of c-MYC, CCND1, CDK4, and CDK6. D: Statistical analysis results of the protein expression of c-MYC, CCND1, CDK4, and CDK6. E: Effects of HOE on the tumor histopathology based on IHC analysis of c- MYC, CCND1, CDK4, and CDK6 in vivo (Scale bars = 100 μm). F: Statistical analysis results of E.

Article Snippet: Primary antibodies against α-Tubulin (AC012), CCND1 (A11022), CDK4 (A0366), and CDK6 (A1545) were purchased from ABclonal (Wuhan, China).

Techniques: Western Blot, Expressing, Histopathology, In Vivo

Journal: Journal of pharmaceutical and biomedical analysis

Article Title: Integrated metabolomics and proteomics analyses to reveal anticancer mechanism of hemp oil extract in colorectal cancer.

doi: 10.1016/j.jpba.2024.116379

Figure Lengend Snippet: Fig. 6. Bioinformatic analysis of gene expression differences in CRC and adjacent normal tissues through bulk RNA-seq and single-cell RNA-seq. A: The expression levels of c-MYC, CCND1, CDK4 and CDK6 in CRC and adjacent normal tissues across TCGA. *P < 0.01 between two groups. B: Discovery of cell types across 62 CRC and 36 adjacent normal colon specimens. C: The epithelial cell subcluster was visualized using t-SNE according to the disease ontology label. D: The expression of c- MYC in every epithelial cell. E: A dot plot demonstrates the ratio of cells expressing a specific gene to total cells and the scaled mean expression between two labels in the epithelial cell subcluster. F: A boxplot shows the expression levels of c-MYC, CCND1, CDK4 and CDK6 in two labels. G: Kaplan-Meier estimates of progression-free survival for patients with high expressed and low expressed samples in TCGA-COADREAD.

Article Snippet: Primary antibodies against α-Tubulin (AC012), CCND1 (A11022), CDK4 (A0366), and CDK6 (A1545) were purchased from ABclonal (Wuhan, China).

Techniques: Gene Expression, RNA Sequencing, Expressing

Journal: Scientific reports

Article Title: Dengue virus modulates critical cell cycle regulatory proteins in human megakaryocyte cells.

doi: 10.1038/s41598-025-02640-5

Figure Lengend Snippet: Fig. 4. DENV2 upregulates several cells cycle regulatory gene transcripts in a time course kinetics. QRT- PCR analysis showing DENV2 capsid transcript levels (A), or CDK1 (B), CDK2 (C), CDK4 (D), CDK5 (E), CDK6 (F), CDK8, (G), Cyclin A1 (H), Cyclin B1 (I), Cyclin E (J), Cyclin G1 (K), Cyclin D3 (L), CDC2 (M) or CDC25A (N) gene expression at days 1, 3 and 5 post DENV2-infection. White bars represent uninfected group and black bars denotes DENV2-infected group. QRT-PCR data is shown as independent analysis for several cell cycle genes either present or absent in the microarray analysis. DENV2-loads and mRNA expression levels of cell cycle regulatory genes were normalized to human beta-actin mRNA levels. The asterisk shows significance, and *, or ** indicates, P value from non-paired two tail student’s t-test, shown as less than 0.05, or 0.01, respectively. Not significant values are denoted with ns.

Article Snippet: Rabbit monoclonal/polyclonal antibodies for CDK4 (catalog number A16813), Phospho CDK4 (catalog number AP0593), CDK1 (catalog number A11420), and Cyclin B1 (catalog number A16038) in 1:1000/2500 dilutions were used (antibodies were obtained from Abclonal Biotechnology Company).

Techniques: Quantitative RT-PCR, Gene Expression, Infection, Microarray, Expressing

Journal: Scientific reports

Article Title: Dengue virus modulates critical cell cycle regulatory proteins in human megakaryocyte cells.

doi: 10.1038/s41598-025-02640-5

Figure Lengend Snippet: Fig. 5. DENV2 infection upregulates CDK4, CDK1 and Cyclin B1 transcripts and protein levels. QRT-PCR analysis showing DENV2 capsid transcript levels (A), or CDK4 (B), CDK1 (C) and Cyclin B1 (D) gene expression in DENV2-infected (MOI of 5, at 72 h p.i.) or uninfected MEG-01 cells. DENV2-loads and mRNA expression levels of CDK4, CDK1 and Cyclin B1 genes were normalized to human beta-actin mRNA levels. Y-axis represents levels of transcript and X-axis indicates uninfected or DENV2-infected groups. P value was determined by Student’s two-tail-test is shown. Each circle or square indicates one data value and n = 6 (six independent replicates run in duplicate for QRT-PCR). Immunoblotting analyses (E–J) performed using cell lysates generated from uninfected or DENV2-infected (MOI of 5, at 72 h p.i.) MEG-01 cells are shown. Immunoblot images showing DENV2-capsid protein loads (E), or CDK4 (F), Phospho-CDK4 (G), CDK1 (H), Cyclin B1 (I) and actin (J) protein loads from uninfected or DENV2-infceted samples. Total protein profile images serve as loading controls for respective immunoblots.

Article Snippet: Rabbit monoclonal/polyclonal antibodies for CDK4 (catalog number A16813), Phospho CDK4 (catalog number AP0593), CDK1 (catalog number A11420), and Cyclin B1 (catalog number A16038) in 1:1000/2500 dilutions were used (antibodies were obtained from Abclonal Biotechnology Company).

Techniques: Infection, Quantitative RT-PCR, Gene Expression, Expressing, Western Blot, Generated

Journal: Scientific reports

Article Title: Dengue virus modulates critical cell cycle regulatory proteins in human megakaryocyte cells.

doi: 10.1038/s41598-025-02640-5

Figure Lengend Snippet: Fig. 6. Silencing of CDK4 or CDK1 or Cyclin B1 in MEG-01 cells reduced DENV2 loads and enhanced CDK1 and Cyclin B1 interactions is enhanced upon DENV2 infection. QRT-PCR and immunoblotting analyses showing silencing of CDK4 (A, D) or CDK1 (B, E) or Cyclin B1 (C, F) in MEG-01 cells transfected with either scrambled siRNA or siRNA for respective genes (CDK4, CDK1, and Cyclin B1) for 24 h and followed by DENV2 infection (MOI of 5, at 24 h p.i.). QRT-PCR and immunoblotting analyses is shown for DENV2 capsid gene or protein loads determined upon silencing of CDK4 (D, J), or CDK1 (H, K) or Cyclin B1 (I, L) in MEG-01 cells transfected with either scrambled siRNA or siRNA for respective genes (CDK4, CDK1, and Cyclin B1) for 24 h and followed by DENV2 infection (MOI of 5, at 24 h p.i.). DENV2-loads and mRNA expression levels of CDK4, CDK1 and Cyclin B1 genes were normalized to human beta-actin mRNA levels. Y-axis represents levels of transcripts and X-axis indicates uninfected or DENV2-infected groups. P value determined by student’s t-test is shown. Scrambled siRNA-treated samples serve as negative control. (M) Immunoprecipitation assay performed with MEG-01 cell lysates from uninfected or DENV2-infected sample (MOI of 5, at 72 h p.i.) using Cyclin B1 antibody and detection with CDK1 antibody is shown for interaction between Cyclin B1 and CDK1 upon DENV2 infection. Total protein profile images serve as loading control (in panels D, E, F, J, K, L and M). (N) Schematic representation shown as model to summarize the current findings. DENV2 enters megakaryocyte cells and upregulates CDK4 protein, which eventually may enter the nucleus and facilitate cell progression from G1 to S phase. DENV2 induces the expression of CDK1 and Cyclin B1 and their interactions that may further assist in the progression of megakaryocytes through the G2 phase of the cell cycle into mitosis. Once the megakaryocytes enter the metaphase, DENV2 may reduce the expression of certain molecules, which are inhibitory to the progression of anaphase A. As a result, DENV2 modulates the cell cycle process in megakaryocytes by pushing from one phase to the next and eventually creating a favorable environment for DENV2 to selectively replicate inside megakaryocytes that undergoes polyploidy and endomitosis.

Article Snippet: Rabbit monoclonal/polyclonal antibodies for CDK4 (catalog number A16813), Phospho CDK4 (catalog number AP0593), CDK1 (catalog number A11420), and Cyclin B1 (catalog number A16038) in 1:1000/2500 dilutions were used (antibodies were obtained from Abclonal Biotechnology Company).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Transfection, Expressing, Negative Control, Immunoprecipitation, Control

Journal: Cell Cycle

Article Title: Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus

doi: 10.1080/15384101.2016.1249546

Figure Lengend Snippet: Specificity in the feline species of the antibodies used. Western blot using proteins from HEK293T cells (A, positive control) and Crandel Feline kidney cells (B, CrFK). Anti-cyclinA, anti-Cdk2, anti Cdk4 antibodies label a single band at the expected molecular weight.

Article Snippet: Histology and immunohistochemistry The following primary antibodies were used with different dilutions for light (L) or fluorescence (F) microscopy, without or with amplification (A), and, for some antibodies, after pretreatment with citrate buffer (C) or TRIS EDTA buffer (T); dilutions for western blot (WB) were also different: mouse anti canine parvovirus (Santa Cruz sc-57961, L 1/50); rabbit anti calbindin (Swant, L 1/5000); rabbit anti MAP2 (Abcam ab32454, L 1/500, F 1/100 A, C or T); mouse anti MAP2 (Sigma Aldrich, clone HM-2, M4403, L 1/500, F 1/100 A, C); mouse anti PCNA (Abcam clone PC10, Ab29, L 1/1000, F 1/100, C); rabbit anti PCNA (Santa Cruz, sc-7907, L 1/500); rabbit anti cyclin D1 (Bios bs-0623, L 1/2000, C); rabbit anti cyclin A (Santa Cruz, sc-751, L 1/100, F 1/100 A, C; WB 1/1000); rabbit anti cdk2 (Spring Bioscience, clone SP80, L 1/100, F 1/50 A, C; WB 1/1000); rabbit anti cdk4 (Novus Biologicals, NBP1-31308, L 1/100, T; WB 1/100); mouse anti phospho-histone H2A.X (Merck Millipore, clone JBW301, L 1/400, F 1/50, T); rabbit anti ERK1 (Santa Cruz, sc93, L 1/100).

Techniques: Western Blot, Positive Control, Molecular Weight

Journal: Cell Cycle

Article Title: Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus

doi: 10.1080/15384101.2016.1249546

Figure Lengend Snippet: Immunohistochemical validation in the feline species of the antibodies used. Immunostainings on a feline mammary adenocarcinoma. (a and h) representative examples of negative controls (no primary antibody); (B) anti-cyclin D, (C) anti-cyclin A, (D) anti-PCNA, (E) anti-Cdk4, (F) anti-Cdk2, (G) anti- H2A1. Some tumor cell nuclei are immunostained. Bar = 100µm.

Article Snippet: Histology and immunohistochemistry The following primary antibodies were used with different dilutions for light (L) or fluorescence (F) microscopy, without or with amplification (A), and, for some antibodies, after pretreatment with citrate buffer (C) or TRIS EDTA buffer (T); dilutions for western blot (WB) were also different: mouse anti canine parvovirus (Santa Cruz sc-57961, L 1/50); rabbit anti calbindin (Swant, L 1/5000); rabbit anti MAP2 (Abcam ab32454, L 1/500, F 1/100 A, C or T); mouse anti MAP2 (Sigma Aldrich, clone HM-2, M4403, L 1/500, F 1/100 A, C); mouse anti PCNA (Abcam clone PC10, Ab29, L 1/1000, F 1/100, C); rabbit anti PCNA (Santa Cruz, sc-7907, L 1/500); rabbit anti cyclin D1 (Bios bs-0623, L 1/2000, C); rabbit anti cyclin A (Santa Cruz, sc-751, L 1/100, F 1/100 A, C; WB 1/1000); rabbit anti cdk2 (Spring Bioscience, clone SP80, L 1/100, F 1/50 A, C; WB 1/1000); rabbit anti cdk4 (Novus Biologicals, NBP1-31308, L 1/100, T; WB 1/100); mouse anti phospho-histone H2A.X (Merck Millipore, clone JBW301, L 1/400, F 1/50, T); rabbit anti ERK1 (Santa Cruz, sc93, L 1/100).

Techniques: Immunohistochemical staining, Biomarker Discovery