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Depletion of T-cell intracellular antigen proteins promotes cell proliferation

Raquel Reyes, José Alcalde and José M Izquierdo*

Author Affiliations

Centro de Biología Molecular 'Severo Ochoa', Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, C/Nicolás Cabrera 1, Lab-107, Cantoblanco DP 28049, Madrid, Spain

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Genome Biology 2009, 10:R87  doi:10.1186/gb-2009-10-8-r87

Published: 26 August 2009

Additional files

Additional data file 1:

(a) MvA plots. From left to right: MvA plots derived from high density oligonucleotide arrays to define gene expression profiles from control (C_1-3.CEL files) and TIA-1 (1_1-3.CEL files), TIAR (R_1-3.CEL files) or TIA-1 plus TIAR (1+R_1-3.CEL files)-depleted HeLa cells. This kind of plot is used to diagnose experimental problems between different biological replicates. The ratio between array signal intensities is illustrated on the y-axis (M) and intensity average is represented on the x-axis (A). In this type of plot, if there are no systematic differences between biological replicates, the dot map of each array is concentrated on the axis M = 0. In all cases documented in our experiments, there was no a significant variability between different biological samples. (b) RNA digestion plots. RNA degradation mainly started from the 5'-end and, therefore, a greater loss of signal intensity occurred with the probes that hybridize to the mRNA 5'-end than with complementary probes close to the mRNA 3'-end. RNA degradation plots illustrate mRNA intensity values versus the function of the positions corresponding to the probes in the 5'-3' orientation. Each graphic on the plot illustrates one array and the slope indicates the RNA degradation level corresponding to each hybridized sample. Overall, a higher slope indicates that more hybridized sample was degraded. This kind of plot is likely to detect significant differences between hybridized samples. Our results show that all samples behaved similarly, suggesting that there are no significant differences between biological samples analyzed. (c) Box plots of crude (to the left) and normalized (to the right) data analyzed by the quantile technique. The Data_rma.txt file submitted to the ArrayExpress database contains processed data using the Robust Multichip Average method [28]. Every color indicates a characteristic group of biological samples analyzed.

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Additional data file 2:

List of the significantly up- and down-regulated genes in TIA-1 and TIAR-depleted HeLa cells analyzed by using Integromics™ software [39]. The studied gene parameters are as follow. Affymetrix Probe Set ID: probe-set identifier of the Affymetrix database [42]. Average Fold Change: mean value of fold change, expressed as base 2 logarithm, for each gene between the compared situations. RP Value Up: probability for a specific gene to be up-regulated. Expected UP: expected value for a certain RP Value Up (it represents the average number of times that an equal or better RP value appears in randomized experiments). FDR Up: expected percentage of false positives for up-regulated genes. RP Value Down: probability for a specific gene to be down-regulated. Expected Down: expected value for a certain RP Value Down. FDR Down: expected percentage of false positives for down-regulated genes. Venn Up: shows in which of the three compared situations (control versus TIA-1 (T1), control versus TIAR (T2) or control versus TIA-1 and TIAR (T3)) the gene is up-regulated. Venn Down: shows in which of the three compared situations as above the gene is down-regulated. UniGene ID: identification number of each gene in the UniGene database [43]. Gene Title: gene name. Gene Symbol: acronym of each gene. Chromosomal Location: gene location on the chromosome. Entrez Gene: probe-set identifier in the Entrez Gene database [44]. SwissProt: protein accession number in SwissProt database [45]. OMIM: accession number in Online Mendelian Inheritance in Man, a database for human genes and genetic disorders [46]. GO Biological Process, GO Cellular Component, GO Molecular Function: accession number and description of the assigned GO in biological process, cellular component and molecular function categories [47]. Pathway: accession number and gene pathway assigned in the biologically represented pathways of the GenMAPP database [48]. InterPro: accession number in the InterPro database and description of protein families and domains [49].

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Additional data file 3:

Analyses of KEGG pathways and GO terms corresponding to the biological process category were applied to differentially expressed genes in TIA-1 and TIAR knockdown HeLa cells. Calculated P-values for each pathway or category are shown. All analyses were performed using Integromics™ software [39].

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Additional data file 4:

List of the up- and down-regulated genes in TIA-1-depleted HeLa cells. The analyzed databases were the same as in Additional data file 2.

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Additional data file 5:

List of the up- and down-regulated genes in TIAR-depleted HeLa cells. The analyzed databases were the same as in Additional data file 2.

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Additional data file 6:

(a-h) Genome-wide microarrays were performed with TIA-1 or TIAR-depleted HeLa cells as in Figure 2. (a-d) Graphic pie representations of the distribution of up- (a, b) and down-regulated (c, d) genes (P < 0.01) in TIA-1 (a, c) or TIAR (b, d) knockdown HeLa cells using the biological process category of the GO database. (e-h) Graphic pie representations of the distribution of up- (e, f) and down-regulated (g, h) genes (P < 0.05) in TIA-1 (e, g) or TIAR (f, h) knockdown HeLa cells using the KEGG pathway database. In all cases (a-h), percentages indicat the portion of total genes that are associated with the biological functions and pathways shown.

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Additional data file 7:

Analysis of KEGG pathways and GO terms corresponding to the biological process category was applied to differentially expressed genes in TIA-1 knockdown HeLa cells. Calculated P-values for each pathway or category are shown. All analyses were performed using Integromics™ software [39].

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Additional data file 8:

Analysis of KEGG pathways and GO terms corresponding to the biological process category was applied to differentially expressed genes in TIAR knockdown HeLa cells. Calculated P-values for each pathway or category are shown. All analyses were performed using Integromics™ software [39].

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Additional data file 9:

Biochemical relationships between gene clusters up-regulated (red) and down-regulated (green) as identified by microarray analyses were created using Pathway Studio software (version 6.0) from Ariadne Genomics [40].

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Additional data file 10:

Summary of differentially expressed genes in TIA-1 and TIAR-depleted HeLa cells that function as transcription and/or cell cycle regulators. The set of studied gene parameters are as follow. Gene Symbol: acronym of each gene. Gene Title: gene name. Fold Change: mean value of fold change for each gene between control and TIA-1 and TIAR-depleted HeLa cells. GO Biological Process: accession number and description of the assigned GO in the biological process category [47]. Functions: summary of the more important roles associated with the list of transcription and/or cell-cycle-related genes.

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Additional data file 11:

The primer sequences were designed using the Universal ProbeLibrary Assay Design Center software from Roche [50] and yielded only one amplified product using a BLAST search [51]. The analyzed genes were: APPBP2, Amyloid beta precursor protein binding protein 2; AREG, Amphiregulin; β-actin, Actin, beta; CD24, CD24 molecule; CXCL1, Chemokine (C-X-C motif) ligand 1; CXCL2, Chemokine (C-X-C motif) ligand 2; EREG, Epiregulin; FASTK, Fas-activated serine/threonine kinase; GDF15, Growth differentiation factor 15; IL-1A, Interleukin 1, alpha; IL-6, Interleukin 6; IL-8, Interleukin 8; KYNU, Kynureninase (L-kynurenine hydrolase); MMP2, Matrix metallopeptidase 2; MTFR1, Mitochondrial fission regulator 1; OPN1, Opsin 1 short-wave-sensitive; PAK3, p21 protein (Cdc42/Rac)-activated kinase 3; PTGS2, Prostaglandin-endoperoxide synthase 2; RAB40B, RAB40B, member of the RAS oncogene family; TFDP2, Transcription factor Dp-2; TIA-1, T-cell intracellular antigen 1; TIAR, TIA-1 related protein; TIMP2, TIMP metallopeptidase inhibitor 2; TMSL8, Thymosin like-8; TNFSF10, Tumor necrosis factor superfamily, member 10; and UCP2, Mitochondrial uncoupling protein 2.

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