Journal: Cancer medicine

Article Title: Nuclear receptor coactivator 6 (NCoA6) promotes cell proliferation, migration, and invasion in pancreatic cancer.

doi: 10.1002/cam4.6427

Figure Lengend Snippet: FIGURE 3 NCoA6 knockdown decreases the proliferation, cell cycle, and apoptosis of pancreatic cancer cells. CCK-8 assays (A, B) and colony formation assays (C, D) were used to test the proliferation of PDAC cells transfected with NCoA6 shRNAs. The representative images and statistical results of the cell cycle (E, F) and apoptosis (G, H) using flow cytometry. (I, J) Western blotting analysis of p21 and p27 expression in NCoA6-silenced PANC-1 and SW1990 cells. (K, L) Western blotting analysis of CDK4, CDK2, Cyclin D1, Cyclin E1, and Cyclin A2 expression in NCoA6-silenced PANC-1 and SW1990 cells.

Article Snippet: Protein extraction and western blotting were performed as described in our previous research.16 Antibodies against NCoA6 (1:1000), E- cadherin (1:5000), N- cadherin (1:3000), FBW7 (1:1000), CDX2 (1:1000), cyclin- dependent kinase 4 (CDK4, 1:2000), cyclin- dependent kinase 2 (CDK2, 1:5000), Cyclin D1 (1:5000), Cyclin E1 (1:1000), and Cyclin A2 (1:2000) were obtained from Proteintech.

Techniques: Knockdown, CCK-8 Assay, Transfection, Flow Cytometry, Western Blot, Expressing

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet: Cdk1 regulates Cyclin B accumulation in G2 phase (A) Schematic. Cdk1 and Plk1 activities that induce mitosis are detected at the S/G2 border and increase gradually throughout G2 phase. (B) Example of unsynchronized U2OS Cyclin B1-YFP cells growing on micropatterns. Lighter colors indicate higher Cyclin B1-YFP fluorescence. Arrows indicate addition of DMSO or inhibitors. Time lapse 30 min. Scale bar 20 μm. (C) Schematic of the in silico synchronization setup. DMSO-treated cells are synchronized in mitosis (top). The resulting Cyclin B1-YFP fluorescence curve (top right) is used to fit Cyclin B1-YFP fluorescence of individual cells before kinase inhibitor treatment (bottom). (D) Quantification of Cyclin B1-YFP fluorescence of cells growing on micropatterns, treated with DMSO or indicated kinase inhibitors after in silico synchronization as in (B) and (C). Graph shows average and standard error of Cyclin B1-YFP fluorescence (At least 15 cells per condition are showed. Data are representative of 4 additional independent experiments, except for Plk1 inhibitor that is present in 2 additional independent experiments). Please note that only Cyclin B1-YFP fluorescence after kinase inhibitor addition is plotted. (E) Quantification of Cyclin B1, Aurora A, and Aurora B immunofluorescence in 4N U2OS Cdk1as cells after 2 h treatment with 1NMPP1. At least 420 cells per condition are showed. Data are representative of 3 independent experiments. ∗∗∗ p < 0.001, using Student’s t test. Interquartile range and median values are indicated within violin plots.

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Fluorescence, In Silico, Immunofluorescence

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet: Cdk1 regulates transcription of mitotic factors (A) Quantification of Cyclin B1 immunofluorescence in 4N U2OS cells treated with Cdk1 inhibitor (RO3306), cycloheximide, or both for 2 h. The G2 population was separated in silico based on DAPI. At least 520 cells per condition are showed. Data are representative of 2 independent experiments; ∗∗∗ p < 0.001, using ANOVA. Interquartile range and median values are indicated within violin plots. (B) Schematic of the setup for RNA sequencing. HeLa cells were released after double thymidine synchronization. After 4.5 h Cdk1 inhibitor (RO3306) or DMSO was added. After 2 h cells were harvested for RNA sequencing analysis. (C and D) Volcano plots show log2 fold change between treated (RO3306) and non-treated (DMSO) normalized gene expressions (x axis), plotted versus the p value (y axis). Orange circles represent differentially expressed genes, and blue circles represent genes with similar expression (data from 3 independent experiments). (E) Schematic containing a selection of key components involved in direct, inner, and outer feedback regulating Cdk activity. (F) Gene Ontology and p values based on (C).

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Immunofluorescence, In Silico, RNA Sequencing, Expressing, Selection, Activity Assay

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet: Mathematical model of the cell cycle (A) Schematic representation of the mathematical model. (B) Model prediction (red line) of protein level dynamics after parameter estimation based on quantitative immunofluorescence of indicated proteins in U2OS cells from Akopyan et al. (blue dots). Please note that experimental data are only used until mitotic entry. The decrease of protein levels in mitosis is added to the model to mark mitosis upon full activation of Cdk1. (C) Model estimation of selected cell-cycle activities. Model time refers to number of calculation steps with a fixed duration. (D) Model prediction of Cyclin B level dynamics after inhibition of Cdk1, Cdk2, or Plk1.

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Immunofluorescence, Activation Assay, Inhibition

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet: Mitotic duration after Wee1 inhibition depends on when in G2 phase Wee1 inhibitors are added (A) Model prediction of G2 duration after Wee1 inhibition at different time points in G2 phase. Time when Wee1i added same as in (B) and (C). G2 phase starts approximately at model time 740, and 20 model time corresponds approximately to 30 min. How activities change relative to model time is visible in Figure 3 . (B) Model prediction of accumulated FoxM activity at mitotic entry after Wee1 inhibition at different time points in G2 phase. 100% denotes mitotic levels in absence of Wee1 inhibition. (C) Model prediction of Cyclin B level at mitotic entry after Wee1 inhibition at different time points in G2 phase. 100% denotes mitotic levels in absence of Wee1 inhibition. Striped line indicates apparent minimum Cyclin B levels at mitotic entry. (D and E) U2OS Cyclin B1-YFP cells were monitored by time-lapse microscopy upon addition of Wee1 inhibitors. Three different inhibitors were used: MK1775 (1 μM), PD0166285 (1 μM), and PD407824 (5 μM). (D) Duration of mitosis (x axis) is plotted versus Cyclin B1-YFP level at mitotic entry (y axis). 100% denotes median mitotic levels in absence of Wee1 inhibition. (E) Duration of mitosis (y axis) is plotted versus estimated time before mitosis should Wee1 inhibitors have not been added (x axis). The estimate is based on Cyclin B1-YFP accumulation of control cells in the same experiment ( Figures S3 E and S3F). 31–70 cells per condition are showed. The data are representative of 3 independent experiments.

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Inhibition, Activity Assay, Time-lapse Microscopy, Control

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet: Wee1 inhibition in G2 phase leads to a de-coupling of Cdk1 and Plk1 activities (A) Model prediction of Plk1 activity at mitotic entry after Wee1 inhibition at different time points in G2 phase. 100% denotes mitotic levels in absence of Wee1 inhibition. G2 phase starts approximately at model time 740, and 20 model time corresponds approximately to 30 min. How activities change relative to model time is visible in Figure 3 . (B) Model prediction of Cyclin B level, Plk1 level, Plk1 activity, and Cdk1 activity after inhibition of Wee1 at different time points in G2 phase. The dotted line in each graph represents the levels at mitotic entry when Wee1 is not artificially inhibited. Mit indicates mitosis. (C) Flow cytometry analysis of Plk1 levels and Plk1-mediated phosphorylation of TCTP (pTCTP) in mitotic U2OS cells. STLC (10 μM), to block cells in mitosis, was added with or without Wee1i for 2 h before harvest. Graph shows mitotic cells, gated as in Figure S5 A. Wee1i, cells treated with the Wee1 inhibitor MK1775 (1 μM) together with STLC; DMSO, cells treated with DMSO together with STLC. At least 2,000 mitotic cells were quantified per condition in two independent experiments.

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Inhibition, Activity Assay, Flow Cytometry, Phospho-proteomics, Blocking Assay

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Recombinant, Gene Expression, RNA Sequencing, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: Silencing of LINC01013 inhibited hypoxia-induced hPASMC proliferation and inflammation. A , B hPASMC proliferation was determined by CCK8 and EdU incorporation assays ( n = 6). EdU (red), DAPI (blue), scale bar, 50 μm. C Western blotting analysis of PCNA, cyclin A and cyclin D in hPASMCs ( n = 6). D RT‒qPCR analysis showed the mRNA levels of TNF-α and IL-6 after LINC01013 knockdown, β-actin was used as a internal reference gene ( n = 6). E Western blotting analysis of TNF-α and IL-6 in hPASMCs. ( n = 6). All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; si, siRNA

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Western Blot, Knockdown, Negative Control

Journal: bioRxiv

Article Title: Cyclin binding Cy motifs have multiple activities in the initiation of DNA replication

doi: 10.1101/681668

Figure Lengend Snippet: (A) Schematic of dynamic expression pattern of human and yeast ORC1 and CDC6 proteins across the cell division cycle with fluctuating levels of Cyclin-CDK kinases. (B) Immunoprecipitation of ORC1 from asynchronous U2OS (left panel) and HeLa (right panel) cell lysates showing interactions with CDC6, ORC3, ORC4, Cyclin A and SKP2 proteins. (C) Dynamic expression profile of pre-RC proteins such as ORC1, CDC6 and CDT1 along with cell cycle marker proteins (Cyclin E, Cyclin A, SETD8 and histone H3S10 phosphorylation) were studied by immunoblotting of extracts from double thymidine block and released synchronized HeLa cells. Flow cytometry profile of DNA content of synchronized HeLa cells at the indicated time in hours after release and corresponding phase of the cell cycle is indicated. (D-E) Double thymidine synchronized HeLa cell lysate at different time points were immunoprecipitated either with an ORC1 antibody (D) or a CDC6 antibody (E) and immunoblotted as indicated. The input and IgG IP denote loading control and mock IP in the experiment, respectively.

Article Snippet: For immunoprecipitations, the following antibodies were used: mouse monoclonal anti-GFP antibody (A11120; Invitrogen), rabbit polyclonal anti-CDC6 antibody (CS1881, CSHL facility), rabbit polyclonal anti-Cyclin A antibody (A305-254A; Bethyl), mouse monoclonal ORC1 78-1-172 ( ) and mouse monoclonal anti-T7 antibody (CSHL Facility).

Techniques: Expressing, Immunoprecipitation, Marker, Western Blot, Blocking Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Cyclin binding Cy motifs have multiple activities in the initiation of DNA replication

doi: 10.1101/681668

Figure Lengend Snippet: (A) Purified MBP-ORC subunit proteins bound to α-MBP antibody magnetic beads were incubated with GST-CDC6 protein in an MBP pull-down assay and blotted with anti-GST antibody (bottom panel). The loading of MBP-ORC1 proteins used in pull-down assays is shown in the top panel. MBP protein served as negative control. (B-C) The recombinant GST-CDC6 full length as well as its various deletion mutant proteins bound to α-GST antibody magnetic beads were incubated with purified MBP-ORC1 protein in a GST pull-down assay and blotted with anti-MBP antibody (bottom panel). In the bottom panel of (C), equitable loading of GST-fused internal regions of CDC6 protein deleted from full length context are indicated. GST-C6 denotes GST-CDC6 protein. (D-E) The purified recombinant GST-CDC6 wild type and Cy mutant proteins bound to α-GST antibody magnetic beads were incubated with either MBP-ORC1 (D) or Cyclin A-CDK2 (E) and blotted with anti-MBP and anti-Cyclin A antibodies, respectively. The loading of GST-CDC6 proteins used in pull-down assay is shown in the top panel. GST protein served as negative control. (F) Interaction between purified recombinant full length ORC1 and CDC6 proteins in an MBP pull-down assay. MBP-ORC1 wild type or its Cy mutant protein bound to α-MBP antibody magnetic beads were incubated with either GST-CDC6 WT or Cy mutant proteins and were immunoblotted with anti-CDC6 antibody. MBP protein served as negative control.

Article Snippet: For immunoprecipitations, the following antibodies were used: mouse monoclonal anti-GFP antibody (A11120; Invitrogen), rabbit polyclonal anti-CDC6 antibody (CS1881, CSHL facility), rabbit polyclonal anti-Cyclin A antibody (A305-254A; Bethyl), mouse monoclonal ORC1 78-1-172 ( ) and mouse monoclonal anti-T7 antibody (CSHL Facility).

Techniques: Purification, Magnetic Beads, Incubation, Pull Down Assay, Negative Control, Recombinant, Mutagenesis

Journal: bioRxiv

Article Title: Cyclin binding Cy motifs have multiple activities in the initiation of DNA replication

doi: 10.1101/681668

Figure Lengend Snippet: (A-B) Mapping of CDC6 interaction region in ORC1 protein. MBP-ORC1 WT or its C-terminal deletion mutant proteins (A) or N-terminal deletion mutant proteins (B) bound to α-MBP antibody magnetic beads were incubated with GST-CDC6 protein in an MBP pull-down assay and immunoblotted with anti-CDC6 antibody (bottom panel). The loading of MBP-ORC1 proteins used in pull-down assay is shown in top panel. MBP protein served as negative control. MBP-O1 denotes MBP-ORC1. (C) The GST-CDC6 proteins bound to α-GST antibody magnetic beads were incubated with MBP-ORC1 either in presence or absence of Cyclin E-CDK2 and Cyclin A-CDK2 and blotted with anti-MBP antibody in GST pull-down assay. Each of the reaction contained 1mM ATP. GST protein served as negative control. (D) In a GST pull-down assay, GST-CDC6 protein was incubated with MBP-ORC1 WT or its mutants in presence or absence of Cyclin E-CDK2 (top panel) or Cyclin A-CDK2 (bottom panel) with 1mM ATP. The western blot is probed with anti-MBP antibody and GST protein served as negative control. (E) Mitotic cells of U2OS cells are harvested after nocodazole treatment, and total cell lysates were further incubated with or without lambda protein phosphatase. The samples were used for western blotting with antibodies as indicated. (F) Mitotic cells from stable GFP-ORC1 WT or its mutant cell lines were collected after nocodazole treatment and lysed, and the cell lysates were used for immunoprecipitation with GFP antibody. The samples were further immunoblotted with CDC6 antibody (showing short and long exposure times on autorad X-ray films) and ORC3 antibody. IgG served as negative control.

Article Snippet: For immunoprecipitations, the following antibodies were used: mouse monoclonal anti-GFP antibody (A11120; Invitrogen), rabbit polyclonal anti-CDC6 antibody (CS1881, CSHL facility), rabbit polyclonal anti-Cyclin A antibody (A305-254A; Bethyl), mouse monoclonal ORC1 78-1-172 ( ) and mouse monoclonal anti-T7 antibody (CSHL Facility).

Techniques: Mutagenesis, Magnetic Beads, Incubation, Pull Down Assay, Negative Control, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: Cyclin binding Cy motifs have multiple activities in the initiation of DNA replication

doi: 10.1101/681668

Figure Lengend Snippet: (A-C) Mapping of CDC6 interaction region in ORC1 protein. MBP-ORC1 WT or its various deletion mutant proteins as indicated, bound to α-MBP antibody magnetic beads were further incubated with purified GST-CDC6 protein in MBP pull-down assays and immunoblotted with anti-CDC6 antibody (bottom panels). MBP-ORC1 proteins used in pull-down assays are shown in the top panels. MBP protein served as negative control. (D) In a GST pull-down assay, recombinant GST-CDC6 proteins bound to α-GST antibody magnetic beads were incubated with purified MBP-ORC1 WT or its CDK mutant proteins in presence or absence of Cyclin A-CDK2, in presence of 1mM ATP. The western blot is probed with anti-MBP antibody and GST protein served as negative control. (E) U2OS cells were synchronized in mitosis with different concentrations of Nocodazole as indicated, isolated mitotic cells were released and harvested at different time points into the cell cycle and total cell lysates were analyzed by immunoblotting with different antibodies as indicated.

Article Snippet: For immunoprecipitations, the following antibodies were used: mouse monoclonal anti-GFP antibody (A11120; Invitrogen), rabbit polyclonal anti-CDC6 antibody (CS1881, CSHL facility), rabbit polyclonal anti-Cyclin A antibody (A305-254A; Bethyl), mouse monoclonal ORC1 78-1-172 ( ) and mouse monoclonal anti-T7 antibody (CSHL Facility).

Techniques: Mutagenesis, Magnetic Beads, Incubation, Purification, Negative Control, Pull Down Assay, Recombinant, Western Blot, Isolation

Journal: bioRxiv

Article Title: Cyclin binding Cy motifs have multiple activities in the initiation of DNA replication

doi: 10.1101/681668

Figure Lengend Snippet: (A) ORC1 fragments, MBP-ORC1 100-250 aa WT and MBP-ORC1 100-250 aa A-A mutant proteins bound to α-MBP antibody magnetic beads were incubated with ORC1 230-400-His protein either alone or in presence of Cyclin A-CDK2 with or without 1mM ATP in MBP pull-down assays. The pull-down assays were western blotted with anti-HIS and anti-CDC6 antibodies. (B) Different fragments of ORC1 protein from 230-400aa region were used in MBP pull-down assay. The MBP-ORC1 protein fragments as indicated bound to α-MBP antibody magnetic beads were incubated with either ORC1 180-240-His WT or ORC1 180-240-His A-A mutant proteins and immunoblotted with anti-His antibody. (C) Multiple sequence alignment of basic patch motifs within the 350-400 region of human ORC1 protein and its corresponding homologs across vertebrate species. The two basic motifs (BP4 and BP5) in human ORC1 protein are indicated by red lines above the alignment. Conserved basic residues in the motifs are colored in pink. In multiple sequence alignment, the species representing each of the vertebrate classes are abbreviated as follows: Mammals ( Homo sapiens , Hs; Mus musculus , Mm; Pan troglodytes , Pt), Aves ( Calypte anna , Ca; Gallus gallus , Gg), Reptiles ( Anolis carolinensis , Ac; Python bivittatus , Pb; Gekko japonicus , Gj) and Amphibia ( Xenopus laevis , Xl; Xenopus tropicalis , Xt).

Article Snippet: For immunoprecipitations, the following antibodies were used: mouse monoclonal anti-GFP antibody (A11120; Invitrogen), rabbit polyclonal anti-CDC6 antibody (CS1881, CSHL facility), rabbit polyclonal anti-Cyclin A antibody (A305-254A; Bethyl), mouse monoclonal ORC1 78-1-172 ( ) and mouse monoclonal anti-T7 antibody (CSHL Facility).

Techniques: Mutagenesis, Magnetic Beads, Incubation, Western Blot, Pull Down Assay, Sequencing

Journal: bioRxiv

Article Title: Cyclin binding Cy motifs have multiple activities in the initiation of DNA replication

doi: 10.1101/681668

Figure Lengend Snippet: (A) HeLa cells were synchronized at G1/S using double thymidine block and cells harvested at indicated time points after release from block were lysed. The whole cell extract was immunoprecipitated with anti-Cyclin A antibody and further immunoblotted with indicated antibodies. (B) HEK293 cells were transfected with GFP-ORC1 WT or the indicated Meier-Gorlin mutants. Whole cell lysates were used for immunoprecipitation with anti-GFP antibody. Immunoprecipitates were analyzed with indicated antibodies. (C) The GFP-ORC1 WT, A-A and CDK stable U2OS cell lines were synchronized at G1/S using a double thymidine block and harvested at different time points after release into fresh media. Total lysates were immunoblotted with anti-GFP and anti-ORC3 antibodies.

Article Snippet: For immunoprecipitations, the following antibodies were used: mouse monoclonal anti-GFP antibody (A11120; Invitrogen), rabbit polyclonal anti-CDC6 antibody (CS1881, CSHL facility), rabbit polyclonal anti-Cyclin A antibody (A305-254A; Bethyl), mouse monoclonal ORC1 78-1-172 ( ) and mouse monoclonal anti-T7 antibody (CSHL Facility).

Techniques: Blocking Assay, Immunoprecipitation, Transfection

Journal: bioRxiv

Article Title: Cyclin binding Cy motifs have multiple activities in the initiation of DNA replication

doi: 10.1101/681668

Figure Lengend Snippet: (A) Endogenous ORC1 was immunoprecipitated from asynchronous U2OS (left panel) and HeLa (right panel) cell lysates with anti-ORC1 mouse monoclonal antibody. The immunoprecipitates were immunoblotted with indicated antibodies. (B) Cell lysates of HEK293 cells overexpressing GFP-PP1 isoforms (α, β and γ) were used for immunoprecipitation with anti-GFP antibody and immunoblotted with indicated antibodies. (C-D) Mapping of PP1 interaction region in ORC1 protein. MBP-ORC1 WT or its C-terminal deletion mutant proteins (C) or N-terminal deletion mutant proteins (D) bound to α-MBP antibody magnetic beads were further incubated with purified recombinant GST-PP1α protein in MBP pull-down assays and immunoblotted with anti-PP1α antibody. MBP protein served as negative control. (E) The recombinant MBP-ORC1 full length as well as its various internal deletion mutant proteins as indicated, bound to α-MBP antibody magnetic beads, were incubated with purified GST-PP1α protein in MBP pull-down assays and blotted with anti-PP1α antibody (top panel). The internal regions of MBP-ORC1 protein is deleted in context of full length. Loading of MBP-fused proteins used in pull-down assay is shown in bottom panel. (F) In MBP pull-down assays, MBP-ORC1 WT or its mutant proteins as indicated, bound to α-MBP antibody magnetic beads were incubated with GST-PP1α either alone or in presence of Cyclin E-CDK2 or Cyclin A-CDK2 kinases with 1mM ATP. The western blot is probed with anti-PP1α antibody and MBP protein served as negative control. (G) Stability analysis of endogenous proteins was performed in U2OS cells by cycloheximide chase. Exponentially growing U2OS cells were treated with 25ug/ml of cycloheximide for indicated times. Total cell lysates were immunoblotted with for detection of endogenous levels of indicated proteins. (H) Asynchronous growing U2OS cells were treated with 10ug/ml of roscovitine for indicated times. Total cell lysates were immunoblotted with indicated antibodies.

Article Snippet: For immunoprecipitations, the following antibodies were used: mouse monoclonal anti-GFP antibody (A11120; Invitrogen), rabbit polyclonal anti-CDC6 antibody (CS1881, CSHL facility), rabbit polyclonal anti-Cyclin A antibody (A305-254A; Bethyl), mouse monoclonal ORC1 78-1-172 ( ) and mouse monoclonal anti-T7 antibody (CSHL Facility).

Techniques: Immunoprecipitation, Mutagenesis, Magnetic Beads, Incubation, Purification, Recombinant, Negative Control, Pull Down Assay, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: MiR-29c-3p Suppresses the Migration, Invasion and Cell Cycle in Esophageal Carcinoma via CCNA2/p53 Axis

doi: 10.3389/fbioe.2020.00075

Figure Lengend Snippet: Primer sequence.

Article Snippet: In order to investigate the underlying mechanism of CCNA2 on EC cells, cells were classified into four groups: si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβand si-CCNA2 + PFTβ groups [PFTβ, p53 inhibitor, HY-16702, MedChemExpress, 10 μM ( )]. qRT-PCR was firstly performed to test CCNA2 level in each group, finding that CCNA2 was markedly decreased in cells transfected with si-CCNA2 + DMSO ( ).

Techniques: Sequencing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: MiR-29c-3p Suppresses the Migration, Invasion and Cell Cycle in Esophageal Carcinoma via CCNA2/p53 Axis

doi: 10.3389/fbioe.2020.00075

Figure Lengend Snippet: MiR-29c-3p is decreased in EC tissues accompanied by low survival rate and associated with the increase of CCNA2. TCGA database was utilized to access expression data of miRNAs and mRNAs of ESCA, and (A) the results of differential analysis were plotted in Volcano plots, with red representing high expression and green representing low expression. In panel (B) , miR-29c-3p level in EC tissues were determined as shown in a box plot. (C) Survival analysis of miR-29c-3p in TCGA-ESCA dataset was performed, with the red line as high expression and blue line as low expression. In panel (D) , Venn diagram was made to find the candidate targets of miR-29c-3p, acquiring 10 DEmRNAs. In panel (E) , correlation analysis was conducted between miR-29c-3p and CCNA2 (–0.57) as plotted in a heat map. In panel (F) , CCNA2 expression in EC cells was examined. Clinical tissue samples were used to further explore the (G) expression of miR-29c-3p and CCNA2 mRNA in EC tissues by qRT-PCR, (H) the protein level of CCNA2 (P1, P2, P3 referred to three EC samples) via Western blot and (I) the correlation between miR-29c-3p and CCNA2. * P < 0.05.

Article Snippet: In order to investigate the underlying mechanism of CCNA2 on EC cells, cells were classified into four groups: si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβand si-CCNA2 + PFTβ groups [PFTβ, p53 inhibitor, HY-16702, MedChemExpress, 10 μM ( )]. qRT-PCR was firstly performed to test CCNA2 level in each group, finding that CCNA2 was markedly decreased in cells transfected with si-CCNA2 + DMSO ( ).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: MiR-29c-3p Suppresses the Migration, Invasion and Cell Cycle in Esophageal Carcinoma via CCNA2/p53 Axis

doi: 10.3389/fbioe.2020.00075

Figure Lengend Snippet: MiR-29c-3p targets CCNA2 and inhibits its expression. Targeted binding sites of miR-29c-3p and CCNA2 were predicted before as shown in panel (A) . To investigate their targeted relationship, (B) dual-luciferase assay was performed to confirm their targeted binding, and (C) RIP was conducted to describe the effect of miR-29c-3p on CCNA2. Moreover, (D,E) qRT-PCR and Western blot were carried out to determine CCNA2 expression in mRNA and protein levels in miR-29c-3p mimic transfected cells, so as to further verify such relationship. * P < 0.05.

Article Snippet: In order to investigate the underlying mechanism of CCNA2 on EC cells, cells were classified into four groups: si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβand si-CCNA2 + PFTβ groups [PFTβ, p53 inhibitor, HY-16702, MedChemExpress, 10 μM ( )]. qRT-PCR was firstly performed to test CCNA2 level in each group, finding that CCNA2 was markedly decreased in cells transfected with si-CCNA2 + DMSO ( ).

Techniques: Expressing, Binding Assay, Luciferase, Quantitative RT-PCR, Western Blot, Transfection

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: MiR-29c-3p Suppresses the Migration, Invasion and Cell Cycle in Esophageal Carcinoma via CCNA2/p53 Axis

doi: 10.3389/fbioe.2020.00075

Figure Lengend Snippet: CCNA2 silencing regulates the migration, invasion and cell cycle in EC by promoting p53 signaling pathway. si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβ and si-CCNA2 + PFTβ were transfected into cells. qRT-PCR and Western blot were conducted to determine (A) the CCNA2 mRNA and (B) protein levels of CCNA2 as well as p53. MTT, Transwell, and flow cytometry were performed to investigate the effects of silencing CCNA2 on EC cell activities, including (C) cell viability, (D) migration and invasion, (E) cell cycle. * P < 0.05.

Article Snippet: In order to investigate the underlying mechanism of CCNA2 on EC cells, cells were classified into four groups: si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβand si-CCNA2 + PFTβ groups [PFTβ, p53 inhibitor, HY-16702, MedChemExpress, 10 μM ( )]. qRT-PCR was firstly performed to test CCNA2 level in each group, finding that CCNA2 was markedly decreased in cells transfected with si-CCNA2 + DMSO ( ).

Techniques: Migration, Transfection, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: MiR-29c-3p Suppresses the Migration, Invasion and Cell Cycle in Esophageal Carcinoma via CCNA2/p53 Axis

doi: 10.3389/fbioe.2020.00075

Figure Lengend Snippet: MiR-29c-3p mediates the migration, invasion and cell cycle in EC via CCNA2/p53 axis. Cells were treated with inhibitor NC + si-NC, inhibitor NC + si-CCNA2, miR-29c-3p inhibitor + si-NC and miR-29c-3p inhibitor + si-CCNA2, and then harvested for (A) Western blot to detect the protein levels of CCNA2 and p53. (B) MTT was performed to test cell viability, (C) Transwell was conducted to assay the ability of cell migration and invasion, and (D) flow cytometry was carried out to determine the effect of miR-29c-3p on cell cycle. * P < 0.05.

Article Snippet: In order to investigate the underlying mechanism of CCNA2 on EC cells, cells were classified into four groups: si-NC + DMSO, si-CCNA2 + DMSO, si-NC + PFTβand si-CCNA2 + PFTβ groups [PFTβ, p53 inhibitor, HY-16702, MedChemExpress, 10 μM ( )]. qRT-PCR was firstly performed to test CCNA2 level in each group, finding that CCNA2 was markedly decreased in cells transfected with si-CCNA2 + DMSO ( ).

Techniques: Migration, Western Blot, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Multi-omics analysis and experimental validation of the value of monocyte-associated features in prostate cancer prognosis and immunotherapy

doi: 10.3389/fimmu.2024.1426474

Figure Lengend Snippet: CCNA2 identified as the best prognostic gene among monocyte-associated genes. (A, B) Functional analysis of monocyte-related prognostic genes. (C, D) GBM and RandomForest algorithms to screen key prognostic genes in the TCGA-PRAD dataset. (E, F) GBM and RandomForest algorithms to screen key prognostic genes in the GSE16560 dataset. (G, H) KM curves of CCNA2 and ACSM3 in the TCGA-PRAD dataset. (I, J) KM curves of CCNA2 and ACSM3 in the GSE16560 dataset.

Article Snippet: Next, CCNA2 antibody (BOSTER, PB9424) was applied dropwise to cover the tissue chip, which was left at room temperature for 2 hours.

Techniques: Functional Assay

Journal: Frontiers in Immunology

Article Title: Multi-omics analysis and experimental validation of the value of monocyte-associated features in prostate cancer prognosis and immunotherapy

doi: 10.3389/fimmu.2024.1426474

Figure Lengend Snippet: CCNA2 positively correlates with monocyte infiltration levels. (A) Correlation analysis of CCNA2 and monocyte infiltration levels. (B, C) Analysis of CCNA2 correlation with monocyte markers. (D) Mendelian randomization analysis of high HLA-DR expressing monocytes in relation to prostate cancer. (E–I) Single-cell analysis of the correlation between CCNA2 and immune cell infiltration.

Article Snippet: Next, CCNA2 antibody (BOSTER, PB9424) was applied dropwise to cover the tissue chip, which was left at room temperature for 2 hours.

Techniques: Expressing, Single-cell Analysis

Journal: Frontiers in Immunology

Article Title: Multi-omics analysis and experimental validation of the value of monocyte-associated features in prostate cancer prognosis and immunotherapy

doi: 10.3389/fimmu.2024.1426474

Figure Lengend Snippet: Functional analysis of CCNA2 in PRAD. (A) KEGG analysis of CCNA2 in PRAD. (B–J) GSEA analysis of CCNA2 in PRAD.

Article Snippet: Next, CCNA2 antibody (BOSTER, PB9424) was applied dropwise to cover the tissue chip, which was left at room temperature for 2 hours.

Techniques: Functional Assay

Journal: Frontiers in Immunology

Article Title: Multi-omics analysis and experimental validation of the value of monocyte-associated features in prostate cancer prognosis and immunotherapy

doi: 10.3389/fimmu.2024.1426474

Figure Lengend Snippet: CCNA2 has a high binding capacity to PRAD-targeted drugs. (A) Analysis of the binding capacity of CCNA2 to PD1 inhibitors. (B) Analysis of the binding capacity of CCNA2 to bicalutamide. (C) Analysis of the binding capacity of CCNA2 to enzalutamide. (D) Analysis of the binding capacity of CCNA2 to abiraterone.

Article Snippet: Next, CCNA2 antibody (BOSTER, PB9424) was applied dropwise to cover the tissue chip, which was left at room temperature for 2 hours.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Multi-omics analysis and experimental validation of the value of monocyte-associated features in prostate cancer prognosis and immunotherapy

doi: 10.3389/fimmu.2024.1426474

Figure Lengend Snippet: CCNA2 is highly expressed in PRAD and is associated with poor patient prognosis. (A, B) Differential expression of CCNA2 in PRAD. (C) Diagnostic predictive value of CCNA2 in PRAD. (D) KM curve of overall survival of CCNA2 in PRAD. (E) Prognostic predictive value of CCNA2 in PRAD.

Article Snippet: Next, CCNA2 antibody (BOSTER, PB9424) was applied dropwise to cover the tissue chip, which was left at room temperature for 2 hours.

Techniques: Quantitative Proteomics, Diagnostic Assay