Table 1.
Significant (q≤0.05) differentially-expressed Tier-1 proteins from mass-spectrometry analysis of PRL-1-overexpressing HEK293 cells.
Table 2.
Significant (q≤0.10) differentially expressing mRNA signals from microarray analysis of PRL-1 overexpressing HEK293 cells.
Table 3.
Significant (q≤0.05) differentially expressing mRNA transcripts from qRT-PCR analysis of PRL-1 overexpressing HEK293 cells.
Figure 1.
Cumulative distributions of mRNA expression levels for microarray probesets.
The cumulative distributions of the expression levels of mRNA probesets that were associated with the coding genes of detected and non-detected proteins are respectively shown in blue and red for the empty-vector (EV) group, and in green and yellow for the PRL-1-overexpressing (P1) group. These data demonstrate that the mean mRNA expression level was approximately 4-fold higher for transcripts corresponding to proteins that were detected (red and yellow) in the proteomic analysis compared to those transcripts where proteins were not detected (blue and green) in the proteomic analysis.
Figure 2.
Volcano plot of significant (q≤0.10) differentially-expressed proteins integrated with changes in corresponding mRNA signals
. The dot (•) symbols represent the Tier-1 proteins that were observed to be differentially expressed under PRL-1-overexpressing conditions in HEK293 cells. These protein data are plotted along the X- and Y-axes according to the log of the protein expression ratio and FDR-corrected significance respectively. A positive log2(protein ratio) value indicates an up-regulation of protein expression under PRL-1-overexpressing conditions as compared to controls, while a negative value indicates down-regulation of protein expression. Each protein's corresponding mRNA data is represented by a colored circle around that protein's dot symbol. Each probeset in the microarray experiment that was 1) mapped to a plotted protein's coding gene and was 2) differentially expressed with a significance of p≤0.20 is represented by a colored region. An asterisk (*) indicates an mRNA signal with a significance of p≤0.05. In cases where multiple detected probesets were mapped to the same protein's coding gene, the colored circle is divided into sectors according to the relative contribution that each probeset had to the total mRNA signal. Yellow colors represent an up-regulation of mRNA expression and blue colors indicate a down-regulation at the mRNA level. FC = fold change; FDR = false discovery rate.
Figure 3.
Fold-enrichment of the membership in Gene Ontology categories for differentially expressed Tier-1 proteins under PRL-1 overexpressing conditions in HEK293 cells.
The list of 172 Tier-1 proteins that were differentially expressed with a significance of q≤0.5 was submitted to the DAVID server as described in the Methods. For each cluster in the output and for each of the 3 GO classes (Biological Process, Cellular Component, and Molecular Function), the GO term with the highest fold-enrichment and an FDR-corrected significance by Fisher's exact test of q≤0.25 was selected to represent that cluster and represented here as a vertical bar. The author-ascribed description of the cluster itself is appended in parentheses to each GO term bar label on the horizontal axis. The black bar height represents the total population of proteins in a given GO term category. The white, blue, and yellow bar heights represent the number of differentially expressed Tier-1 proteins from the experiment in a given GO term category. The number shown on the bar is the fold-enrichment for a given GO term category, and the stars following each number represent the FDR-corrected significance of that fold-enrichment (no stars for 0.25≥ q>0.1, * for q≤0.1, ** for q≤0.01, *** for q≤0.001). The fold-enrichment number was calculated as (number of Tier 1 proteins in the GO category/number of Tier 1 proteins)/(number of proteins in the GO category/number of proteins in the GO class). GO = gene ontology; FDR = false discovery rate
Figure 4.
Fold-enrichment of the membership in Gene Ontology categories for differentially expressed mRNA probesets under PRL-1 overexpressing conditions in HEK293 cells
. The list of 2438 mRNA probesets that were differentially expressed with a significance of q≤0.5 was submitted to the DAVID server as described in the Methods. For each cluster in the output and for each of the 3 GO classes (Biological Process, Cellular Component, and Molecular Function), the GO term with the highest fold-enrichment and an FDR-corrected significance by Fisher's exact test of q≤0.25 was selected to represent that cluster and represented here as a vertical bar. The author-ascribed description of the cluster itself is appended in parentheses to each GO term bar label on the horizontal axis. The black bar height represents the total population of probesets in a given GO term category. The white, blue, and yellow bar heights represent the number of differentially expressed mRNA probesets from the experiment in a given GO term category. The number shown on the bar is the fold-enrichment for a given GO term category, and the stars following each number represent the FDR-corrected significance of that fold-enrichment (no stars for 0.25≥ q>0.1;* for q≤0.1, ** for q≤0.01, *** for q≤0.001). The fold-enrichment number is calculated as (number of Tier 1 proteins in the GO category/number of Tier 1 proteins)/(number of proteins in the GO category/number of proteins in the GO class). GO = gene ontology; FDR = false discovery rate.
Figure 5.
Protein changes in the Rho-signaling canonical pathway resulting from PRL-1 overexpression in HEK293 cells.
Selected proteins that conduct signals to remodel the cytoskeleton through RhoA, Rac1, and CDC42 are represented by their coding gene names in a canonical pathway diagram adapted from Ingenuity Pathway Analysis (IPA). The symbols of proteins that were detected in the mass-spectrometry experiment at Tier-1 or Tier-2 levels are colored according to the direction of their fold change (FC) in expression in the PRL-1-transfectants as compared to the empty vector group, with yellow hues indicating an increased quantity of protein and blue hues indicating a decrease. An asterisk (*) indicates that a protein expression change is significant at a level of p≤0.05. Tier-1 proteins are noted with bold font labels. Groups of related or complex-forming proteins are illustrated with double-outlined symbols. Connecting lines with arrowheads indicate an activating, de-activating, or translocating influence, and the absence of an arrowhead indicates a protein-protein binding interaction or group membership. Solid connecting lines show direct interactions while dashed lines show indirect interactions. The known direct interaction of PRL-1 (PTP4A1) with ARHGAP4 is represented here, but indirect connections between PRL-1 and the components of this pathway are not shown.