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Results: 7

1.
Figure 5.

Figure 5. From: Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program.

Altered patterns of RNAP II phosphorylation and elongation factor recruitment on the p21 locus upon activation by DRB treatment. ChIP assays were performed with protein extracts obtained from HCT116 cells before or 8 h after treatment with doxorubicin (0.4 μM) or DRB (50 μM) with antibodies recognizing p53, total RNAP II (RNAP II), CDK9, phospho-Ser-5 CTD (S5P-CTD), phospho-Ser2-CTD (S2P-CTD), and SPT16 (FACT complex). Quantification and replicates were performed as in Figure 2.

Nathan P. Gomes, et al. Genes Dev. 2006 March 1;20(5):601-612.
2.
Figure 3.

Figure 3. From: Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program.

Recruitment of initiation and elongation factors to the p21 locus during activation. ChIP assays were performed with protein extracts obtained from HCT116 cells before or 8 h after doxorubicin treatment with antibodies recognizing TFIIB, TAF1, TBP, NELF-A (NELF complex), SPT5 (DSIF complex), CDK9 (P-TEFb complex), RAP74 (TFIIF complex), CDK7 (TFIIH complex), and SPT16 (FACT complex). Quantification and replicates were performed as in Figure 2. Asterisks indicate the positions of the transcription start site and polyadenylation signal.

Nathan P. Gomes, et al. Genes Dev. 2006 March 1;20(5):601-612.
3.
Figure 7.

Figure 7. From: Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program.

p53-dependent apoptosis in response to DRB treatment. (A) FACS analysis of cell cycle profile. HCT116 p53+/+ cells were harvested at different times after addition of DRB (50 μM), fixed, and stained with propidium iodide; DNA content was determined by FACS. Pie charts display the percentage of cells in each stage of the cell cycle. (B) Apoptotic index assay. Cells were treated and harvested as in A, stained without fixation with annexin V and propidium iodide (PI), and analyzed by FACS. Numbers indicate the percentage of cells in each quadrant.

Nathan P. Gomes, et al. Genes Dev. 2006 March 1;20(5):601-612.
4.
Figure 2.

Figure 2. From: Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program.

Distribution of RNAP II and its phospho-isoforms on the p21 gene locus before and during p53-dependent transcriptional activation. ChIP assays were performed with protein extracts obtained from HCT116 p53+/+ cells before or 8 h after doxorubicin treatment with antibodies recognizing the p53 transactivation domain (p53), total RNAP II (RNAP II), or phosphorylated isoforms of Ser5 and Ser2 of the RNAP II CTD (S5P-CTD, S2P-CTD). ChIP-enriched DNA was quantified by real-time PCR using the indicated amplicons. Values are expressed as percentage of input DNA immunoprecipitated. The results shown are the average of at least eight independent PCRs from four separate immunoprecipitations from two independent cell cultures. All standard deviations are <15%. Asterisks indicate the positions of the transcription start site and polyadenylation signal.

Nathan P. Gomes, et al. Genes Dev. 2006 March 1;20(5):601-612.
5.
Figure 4.

Figure 4. From: Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program.

Activation of the p53 pathway by DRB-mediated inhibition of global transcription. (A) Western blot showing the ratio of phosphorylated (IIo) to unphosphorylated (IIa) RNAP II in HCT116 cells treated with doxorubicin (0.4 μM) or DRB (50 μM). The same blot was also probed with p53 and actin antibodies. (B) Real-time PCR analysis of gene expression in response to doxorubicin or DRB treatment. HCT116 cells of different p53 status were harvested at the indicated times after treatment with doxorubicin (0.4 μM) or DRB (50 μM), total RNA was isolated, and RT–PCR was performed with primers specific to the indicated mRNAs. Values were normalized to those of 18S rRNA and are expressed as fold induction over untreated p53+/+ cells. HPRT and SDHA are housekeeping, non-p53 target genes. Results shown are the average of eight different PCRs from four cDNA preparations of two different RNA extractions.

Nathan P. Gomes, et al. Genes Dev. 2006 March 1;20(5):601-612.
6.
Figure 6.

Figure 6. From: Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program.

Analysis of 3′ RNA processing of p21 mRNA. (A) Western blot showing p21 protein accumulation in HCT116 cells in response to doxorubicin (0.4 μM) or DRB treatment (50 μM). (B) PCR analysis of p21 mRNA 3′ cleavage. Sequence of the 3′-end region of the p21 locus showing the location of the primers used as well as the polyadenylation signal (AATAAA) and the cleavage site (asterisk) as predicted by cDNA cloning (GenBank accession no. NM_000389). PCR was performed using total genomic DNA (gDNA) or cDNA from HCT116 p53+/+ cells before or 16 h after treatment with doxorubicin or DRB. (M) Molecular weight marker. (C) Real-time PCR analysis of p21 mRNA polyadenylation. Total and poly(A)+ RNA were prepared from HCT116 p53+/+ cells before and 16 h after DRB treatment. Equal mass of either preparation was used as template in RT–PCR reactions with primers specific to p21 or HPRT mRNAs. Relative quantification was achieved using standard curves of known amounts of total cDNA. The results shown are the average of six PCRs from two different RNA extractions.

Nathan P. Gomes, et al. Genes Dev. 2006 March 1;20(5):601-612.
7.
Figure 1.

Figure 1. From: Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program.

Kinetics of activation of the p53–p21 axis in HCT116 cells in response to DNA damage. (A) Western blot showing accumulation of p53 and p21 proteins upon doxorubicin treatment (0.4 μM). Nucleolin was used as a loading control. (B) Real-time RT–PCR analysis of p21 mRNA accumulation. Values were normalized to those obtained for 18S ribosomal RNA, and are expressed as fold induction over untreated HCT116 p53+/+ cells. (C) FACS analysis of cell cycle profile. HCT116 p53+/+ cells were harvested at indicated times after addition of doxorubicin, and their DNA content was determined by FACS. Pie charts display the percentage of cells in each stage of the cell cycle. (D) Linear up-to-scale map of the p21 locus showing the location of p53-binding sites (p53BS1 and p53BS2), the transcription start site (+1), exons and introns, the start codon (ATG), the stop codon (TAA), and the polyadenylation signal (AATAAA). The location of 20 amplicons used in real-time PCR quantification of ChIP-enriched DNA is also shown. Numbers indicate the relative position of the central base pair of the amplicon relative to the transcription start site. Amplicon sizes range between 50 and 75 bp.

Nathan P. Gomes, et al. Genes Dev. 2006 March 1;20(5):601-612.

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