Figure 3

Endogenous Bcl-xS expression orchestrates a cellular gene expression program linked to the protein synthesis machinery. (A) Transcriptome analysis following Bcl-x SSO in HeLa cells. Expression levels of mRNAs from Bcl-x AS-treated cells were compared to control oligonucleotide-treated HeLa samples 18 hours post SSO transfection by genome-wide microarray analysis using three independent biologic replicates each. The absolute number of probes detecting statistically significant (p < 0.05) up- or down-regulation following splice-site selection is shown to the left of the bar; the maximum positive or negative logarithmic (base two) fold-change is shown to the right. The black gradient indicates positive, and the white gradient negative fold changes in expression. (B) The relative, normalized, averaged chemiluminescence signal intensities for the least repressed (C3orf1), the least induced (GNA11), the most repressed ("-"; no annotation information available), and the most induced (MGC48628) gene when comparing the Bcl-x AS oligonucleotide (black bars) to the control AS oligonucleotide (grey bars) at a statistical significance threshold of p < 0.05 as well as the standard deviations over those signals are compared. (C) Specific biologic processes and molecular functions are statistically significantly (p < 0.01) overrepresented in the Bcl-xS-transcriptome. "Count" indicates the number of probes within the Bcl-x dataset that can be attributed to the corresponding ontology term, "Expected" is the number of probes one would expect to find for a given ontology term in a random dataset of identical size. (D) Pie-chart analysis of genes down-regulated by endogenous Bcl-xS SSO illustrating the number of genes associated with the ontology term "molecular function: Ribosomal Protein" (grey), and "molecular function: Nucleic Acid Binding" (black) as a fraction of all genes down-regulated by endogenous Bcl-xS SSO. (E) Verification of gene expression changes by quantitative real-time RT-PCR. HeLa cells were transfected with antisense oligonucleotides that induce endogenous Bcl-xS expression. 18 hours post-transfection total RNA was isolated from cells and expression level changes were analyzed by quantitative real-time PCR (grey bars) and compared with changes in expression from microarray measurements (black bars) as logarithmic base-two fold changes. Error bars indicate standard deviation of three independent transfections. The Pearson correlation coefficient calculated for the correlation between the microarray data and the qPCR is 0.872.