Zambon et al. 10.1073/pnas.0503363102. |
Supporting Table 1
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Supporting Figure 6
Fig. 6. Correlation of fold change as determined by using microarrays and quantitative PCR for 15 genes at three time points (n = 45).
Fig. 7.
Growth kinetics of WT and Kin– S49 cells. (A) Growth curves: Three batches of each cell line were grown under the same conditions. Cultures were initiated at a density of 2.5 × 105 cells per ml and grown for up to 72 h. Total cell number was determined with a Z2 Coulter counter (Beckman) and cell viability was determined by trypan blue staining. Each time point measurement was repeated at least twice. (B) Effect of 8-CPT-cAMP on cell growth: For each cell line, WT and Kin– S49 cells were treated with 8-CPT-cAMP for the indicated times. Cell number and viability were determined as described for A and flow cytometry was used for DNA and cell-cycle analysis (Right).Fig. 8.
Coordinated down-regulation of translation factors and tRNA synthetases by increased cAMP levels in S49 cells. Gene transcripts are represented by boxes and colored as indicated. The translational control pathway, from GenMAPP.org (1) (www.GenMAPP.org) was developed by Kam Dahlquist (Vassar College, Poughkeepsie, NY).1. Dahlquist, K. D., Salomonis, N., Vranizan, K., Lawlor, S. C. & Conklin, B. R. (2002) Nat. Genet. 31, 19-20.
Fig. 9.
Frequency distributions and statistical significance of conserved CRE sites within 5 kb of transcriptional start sites. (A) Conserved sites, sites that are aligned in human and mouse and located in the 21-bp window of alignment conserved at ³80% level; y axis, frequency of total hits (total number of conserved sites/total number of genes in cluster or comparison); pairwise comparisons, genes in WT S49 cells changed by cAMP (P < 0.06 fold >50%); All, all genes on the array (negative control). (B) Statistical significance (P value) of enrichment of conserved CRE sites upstream of genes in each cluster. P values are calculated by assuming a binomial distribution B(n, K/N) for the number of conserved CRE sites in a cluster of coexpressed transcripts. K, size of the cluster; N, number of aligned upstream regions in the whole genome; n, number of whole-genome conserved CRE sites detected in the N alignments.Fig. 10.
Distribution of conserved CRE sites relative to the transcriptional start (Tx) site for all aligned upstream regions in the genome. Distance is measured in bp.Fig. 11.
Top 77 scoring CRE-containing genes from Conkright et al. (1) colored with data from WT S49 cells 2 h after cAMP treatment. Boxes represent gene transcripts and are colored as indicated. Numbers to the right of boxes represent fold-changes 2 h after cAMP treatment.1. Conkright, M. D., Guzman, E., Flechner, L., Su, A. I., Hogenesch, J. B. & Montminy, M. (2003) Mol. Cell. 11, 1101-1108.
Fig. 12.
Real-time PCR analysis of cell cycle targets at early timepoints. Replicates are as follows: Icer (n = 4); Gadd45a (n = 4); Cyclin G2 (n = 4); Crem (n = 2); Cyclin E1 (n = 2); Cyclin E2 (n = 2); p27 (n = 2). Error bars: SE.Fig. 13.
Expanded G1/S-phase cell cycle pathway. Gene transcripts are represented by boxes and colored as indicated. Pathway was based on the Reactome (www.reactome.org/) G1/S cell-cycle control (1) and ref. 2.1. Joshi-Tope, G., Vastrik, I., Gopinathrao, G., Matthews, L., Schmidt, E., Gillespie, M., D’Eustachio, P., Jassal, B., Lewis, S., Wu, G., et al. (2003) Cold Spring Harbor Symp. Quant. Biol. 68, 237-243.
2. Hildesheim, J., Bulavin, D. V., Anver, M. R., Alvord, W. G., Hollander, M. C., Vardanian, L. & Fornace, A. J., Jr. (2002) Cancer Res. 62, 7305-7315.