Figure Legends
Figure 1 PDF Gene expression patterns of lymphoma cell line OCI-Ly3 treated with flavopiridol. (a) Hierarchical clustering of OCI-Ly3 cells treated with 60 nM, 300 nM, or 1 mM flavopiridol for 1- or 3-hours. Each column is a single experiment comparing two cDNA populations, treated sample was labeled red (Cy5) and untreated sample was labeled green (Cy3). Each row represents data from a single cDNA microarray spot. The red-to-green (Cy5/Cy3) ratio reflects hybridization to that spot, a measure of relative gene expression; intensity reflects the magnitude of the difference between the samples according to the ratio color scale. Red indicates Cy5/Cy3 ratios >1, green indicates Cy5/Cy3 ratios <1, black indicates no significant change in gene expression, and gray indicates the spot did not meet data selection criteria. These ratios were depicted according to the color scale shown at the bottom. Gene explorer (b) MTT assays. OCI-Ly3 cells were treated with increasing concentration of flavopiridol (0 to 2 mM) for 8-, 24-, or 48-hours. MTT was added to the cells 2 hours before harvesting. The plate was read at 570 nm. The result was shown as percent of control vs. flavopiridol concentration. (c) Hierarchical clustering of OCI-Ly3 cells treated with flavopiridol (1 mM), roscovitine (25 mM), or 9-nitropaullone (2.5 mM) for 1- or 4- hours. The treated samples were labeled red (Cy5) and untreated samples were labeled green (Cy3). Only genes that were downregulated for more than three folds in one or more samples were extracted for clustering. Gene explorer
Figure 2 PDF Similar gene expression patterns of OCI-Ly3 cells treated with flavopiridol, actinomycin D, and DRB. (a) OCI-Ly3 cells were treated with flavopiridol (1 mM), actinomycin D (10 mg/ml), or DRB (100 mM) for 0.5-, 1-, 2-, 4-, and 8-hours. The treated samples were labeled red (Cy5) and untreated samples were labeled green (Cy3). Genes were categorized according to their half-lives by the scheme as described in the methods section. Gene explorer (b) Northern blot analysis of selected genes. (c) Comparison of turnover rate of selected genes from microarray data and Northern blot analysis (percent of control vs. time).
Figure 3 PDF The majority of well-measured genes on the Lymphochip decreased in mRNA abundance with first-order kinetics after transcriptional inhibition. See text for details. Calculated half-lives
Figure 4 PDF Analysis of genes inhibited by flavopiridol. OCI-Ly3 cells were treated with flavopiridol (1 mM) for 0.5-, 2-, 3-, 4-, 6-, or 8-hours. Total RNA was prepared for microarray analysis on Lymphochips with ~17,000 microarray elements. The treated samples were labeled red (Cy5) and untreated samples were labeled green (Cy3). Genes were first categorized according to their half-lives using the scheme as described in the methods section. These genes were then clustered according to their function. (a) Proliferation genes with half-lives less than four hours. The cell cycle phase in which the particular gene expresses is indicated. (b) Anti-apoptotic genes with short half-lives. (c) Percentages of induced or not induced microarray elements as categorized by their half-lives. (d) Induced genes with half-lives less than four hours. See text for details.
Figure 5 PDF Sub-grouping of induced genes of PBMCs. Purified PBMCs were induced with PI for 1-, 3-, 6-, or 24-hours before harvesting for microarray analysis. The induced samples were labeled red (Cy5) and the induced pool (a mix of purified PBMCs induced with PI for 0-, 1-, 3-, 6-, or 24-hours) was labeled green (Cy3). For analyzing the turnover rate of these induced transcripts, purified PBMCs were first induced with PI treatment for 3 hours followed by flavopiridol treatment for 1-, 2-, 4-, or 6-hours before harvesting for microarray analysis. The induced samples treated with flavopiridol were labeled red (Cy5) and induced samples without flavopiridol treatment were labeled green (Cy3). (a) Percentages of genes (induced or not induced by PI) are plotted according to their half-lives. (b) Percentages of genes of certain functional groups (induced or not induced by PI) are plotted according to their half-lives. (c) Microarray data showing highly induced genes with short half-lives of certain functional groups. (d) Microarray data showing half-lives of highly induced chemokines, cytokines, and their receptors.
Figure 6 PDF Percentages of AUUUA-containing transcripts in different half-life groups.
Figure 7 PDF Scheme for determining half-lives in Figure 2, 4, and 5. See Materials and Methods for details.