Journal: FEBS letters

Article Title: Cyclin B1 transcription is enhanced by the p300 coactivator and regulated during the cell cycle by a CHR-dependent repression mechanism.

doi: 10.1016/s0014-5793(03)00028-0

Figure Lengend Snippet: Fig. 1. Cell cycle-dependent expression of chromosomally derived cyclin B1. NIH3T3 mouse ¢broblasts and HFF were serum-starved and re- stimulated by addition of serum to the medium. After 48 h of starvation, the 0 h time point represents cells mostly resting in G0. The following time points give results after serum restimulation. A: Cyclin B1 mRNA expression from NIH3T3 cells as measured by RT-PCR standardised relative to total RNA; averages of two experiments are given. B: FACS analyses of NIH3T3 cell populations at selected time points before and after serum addition. Cells were stained with propidium iodide and measured for DNA staining per cell. C: Expression of cyclin B1 mRNA in HFF cells with total RNA as standard quanti¢ed by RT-PCR. Averages of two experiments are given. D: Cyclin B1 protein expres- sion detected by immunoblotting. L-Actin served as a loading control.

Article Snippet: As negative control served a mixture of 0.5 Wg mouse monoclonal anti-cyclin B1 antibody (GNS1, Santa Cruz Biotechnology) and 0.5 Wg goat polyclonal anti-cyclin B2 antibody (N-20, Santa Cruz Biotechnology).

Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Western Blot, Control

Journal: FEBS letters

Article Title: Cyclin B1 transcription is enhanced by the p300 coactivator and regulated during the cell cycle by a CHR-dependent repression mechanism.

doi: 10.1016/s0014-5793(03)00028-0

Figure Lengend Snippet: Fig. 4. p300 activates transcription from the cyclin B1 gene and binds to its promoter. A: Increasing amounts of plasmids expressing the three subunits of NF-Y (A, B and C) and p300 were cotrans- fected with the cyclin B1 promoter reporter and a Renilla luciferase control plasmid in SaOS-2 cells. The total amount of DNA was held constant in all transfections by adding an irrelevant plasmid. Fire£y luciferase was measured and standardised to Renilla lucifer- ase activity. Expression from the cyclin B1 promoter without co- transfection was set at 100%. Assays from three independent experi- ments with standard deviations are shown. B: ChIP experiments with antibody directed against p300 on the cyclin B1 promoter. The antibodies used in the control lane were directed against non-DNA binding protein. The K-globin gene served as negative control pro- moter.

Article Snippet: As negative control served a mixture of 0.5 Wg mouse monoclonal anti-cyclin B1 antibody (GNS1, Santa Cruz Biotechnology) and 0.5 Wg goat polyclonal anti-cyclin B2 antibody (N-20, Santa Cruz Biotechnology).

Techniques: Expressing, Luciferase, Control, Plasmid Preparation, Transfection, Activity Assay, Cotransfection, Binding Assay, Negative Control

Journal: FEBS letters

Article Title: Cyclin B1 transcription is enhanced by the p300 coactivator and regulated during the cell cycle by a CHR-dependent repression mechanism.

doi: 10.1016/s0014-5793(03)00028-0

Figure Lengend Snippet: Fig. 3. Expression from a cyclin B1-promoter luciferase reporter comparing activity in G2/M versus resting cells. Mutation of the CHR leads to a loss of cell cycle regulation. Luciferase reporter plasmids carrying the human cyclin B1 upstream region together with a Renilla luciferase-expressing control plasmid were transfected into NIH3T3 ¢broblasts. Cells were synchronised by serum starva- tion for 66 h and restimulated. Ratios of luciferase activities from cell lysates 24 h after restimulation to activities in resting cells are given. Averages with standard deviations from six experiments are shown. Renilla luciferase was employed to standardise cyclin B1 ¢re- £y luciferase reporter expression.

Article Snippet: As negative control served a mixture of 0.5 Wg mouse monoclonal anti-cyclin B1 antibody (GNS1, Santa Cruz Biotechnology) and 0.5 Wg goat polyclonal anti-cyclin B2 antibody (N-20, Santa Cruz Biotechnology).

Techniques: Expressing, Luciferase, Activity Assay, Mutagenesis, Control, Plasmid Preparation, Transfection

Journal: FEBS letters

Article Title: Cyclin B1 transcription is enhanced by the p300 coactivator and regulated during the cell cycle by a CHR-dependent repression mechanism.

doi: 10.1016/s0014-5793(03)00028-0

Figure Lengend Snippet: Fig. 4. p300 activates transcription from the cyclin B1 gene and binds to its promoter. A: Increasing amounts of plasmids expressing the three subunits of NF-Y (A, B and C) and p300 were cotrans- fected with the cyclin B1 promoter reporter and a Renilla luciferase control plasmid in SaOS-2 cells. The total amount of DNA was held constant in all transfections by adding an irrelevant plasmid. Fire£y luciferase was measured and standardised to Renilla lucifer- ase activity. Expression from the cyclin B1 promoter without co- transfection was set at 100%. Assays from three independent experi- ments with standard deviations are shown. B: ChIP experiments with antibody directed against p300 on the cyclin B1 promoter. The antibodies used in the control lane were directed against non-DNA binding protein. The K-globin gene served as negative control pro- moter.

Article Snippet: NIH3T3 cells were crosslinked with 1% formaldehyde for 18 h after serum restimulation. p300 protein crosslinks were precipitated using 1 Wg of rabbit polyclonal anti-p300 antibody (C-20, Santa Cruz Biotechnology).

Techniques: Expressing, Luciferase, Control, Plasmid Preparation, Transfection, Activity Assay, Cotransfection, Binding Assay, Negative Control