Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues-1

<p><b>Copyright information:</b></p><p>Taken from "Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues"</p><p>BMC Genomics 2006;7():94-94.</p><p>Published online 26 Apr 2006</p><p>PMCID:PMC1459866.</p><p>Copyright © 2006 Aouacheria et al; licensee BioMed Central Ltd.</p>r-specific or absent in tumors). The results are shown for twelve tissues. The legend is the same as in Figure 1. See additional information for the full data

Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues-3

<p><b>Copyright information:</b></p><p>Taken from "Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues"</p><p>BMC Genomics 2006;7():94-94.</p><p>Published online 26 Apr 2006</p><p>PMCID:PMC1459866.</p><p>Copyright © 2006 Aouacheria et al; licensee BioMed Central Ltd.</p>ngiocellular carcinoma (male, age 46); lane 4: normal liver (male, age 24); lane 5: normal placenta (female, age 24). Bcl-xL immunoreactivity (26 kD) was observed in two out of three liver cancer samples (lanes 1–3). Normal samples (lane 4–5) had no signal for Bcl-xL expression. Note that GADPH protein levels varied between normal tissues and cancer liver specimens and did not correlate with the mRNA levels predicted by the computer-based screen. The expression of tubulin was used as control for equal protein loading

Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues-2

<p><b>Copyright information:</b></p><p>Taken from "Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues"</p><p>BMC Genomics 2006;7():94-94.</p><p>Published online 26 Apr 2006</p><p>PMCID:PMC1459866.</p><p>Copyright © 2006 Aouacheria et al; licensee BioMed Central Ltd.</p>5 studied tissues. The results are shown for fourteen tissues. The color code is the same as in Figure 1. See additional information for the full data

Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues-0

<p><b>Copyright information:</b></p><p>Taken from "Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues"</p><p>BMC Genomics 2006;7():94-94.</p><p>Published online 26 Apr 2006</p><p>PMCID:PMC1459866.</p><p>Copyright © 2006 Aouacheria et al; licensee BioMed Central Ltd.</p>individual normal tissue. The color in each cell reflects the differential expression level of the corresponding gene in a particular tissue. A four color code was used to represent gene induction and repression in cancer libraries (dark green: 'normal-specific', i.e. not expressed in tumor libraries; light green: downregulated in tumor libraries; orange: upregulated in tumor libraries; red: 'tumor-specific'). If there was no significant change in gene expression between normal and tumor libraries or in case of missing/excluded data, the gene was given in a black color. The number inside the colored cells indicates the statistical significance (-value < 0.01 after Bonferroni correction). See additional information for the full data

The Bfl-1-α9 peptide induces mitochondrial permeabilization through a membrane-destabilizing mechanism.

<p>Bfl-1-α9 peptide was incubated for the indicated times with mitochondria at lower concentrations (0.5 µM and 2.5 µM, top panels) and previously used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone-0038620-g004" target="_blank">figure 4</a> (10 µM and 25 µM, bottom panels). The release of cytochrome c and the expression of MitoHsp70 were monitored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone-0038620-g004" target="_blank">figure 4</a>, combined with the detection of the external membrane associated protein hexokinase 1 (HK1) and the matrix-contained protein MnSOD.</p

μ-calpain cleaves Bfl-1 at two major sites in its N-terminus and releases a large C-terminal fragment with cytotoxic activity.

<p>(A) GST-Bfl-1(1–151) was digested with recombinant µ-calpain <i>in vitro</i> and the fragments were separated by SDS-PAGE (left panel). Bands corresponding to cleaved products (arrows) were analyzed by mass spectrometry (MS). A higher concentration of recombinant GST-Bfl-1(1–151) was treated with µ-calpain, products were separated by SDS-PAGE and blotted to PVDF (right panel). A sub-band (asterisk) was excised and subjected to Edman degradation, which indicated that Bfl-1 had been N-terminally cleaved between residues F71 and N72. (B) Schematic representation of the wild type Bfl-1 protein showing the location of the identified μ-calpain cleavage sites (asterisks, upper sequence), of mutant Bfl-1 protein with a 6 aminoacids deletion surrounding the two cleaved residues (Bfl-1DD, middle sequence), and of mutant Bfl-1 in which the region overlapping the first cleavage site was swapped with a structurally homologous region in Bcl2L10 (Bfl-1SD, bottom sequence) (C) Confirmation of the two calpain cleavage sites identified in Bfl-1 using noncleavable mutants. 293T cell lysates expressing GFP-tagged Bfl-1 constructs were exogenously treated with μ-calpain. Lysates containing equal amount of GFP-tagged Bfl-1 proteins were separated by SDS-PAGE and analyzed by western blot with an anti-GFP antibody to detect the full length protein and N-terminal truncated fragments and with a polyclonal anti-Bfl-1 antibody to detect C-terminal truncated fragments. Upper and lower panels represent two independent experiments with different time of exposure. (D) BJAB cells were cultured with or without treatment with TNF/CHX in the presence or absence of the calpain inhibitor ALLN. Lysates were separated by SDS-PAGE and the presence of a cleaved fragment was assayed by western blot using an anti-Bfl-1 antibody. (E) Secondary structure of Bfl-1 in which the nine helices of the protein are represented by boxes along with the different BH domains (left panel).The two cleavage sites mapped in (A) are indicated. A comparison with the previously published µ-calpain cleavage site in Bax is also shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Wood1" target="_blank">[26]</a> (bottom sequence). 3D structure of Bfl-1 (2VM6) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Herman1" target="_blank">[31]</a> and position of the different cleaved sites (red circles) are indicated. The different Bcl-2 Homology domains are colored in yellow (putative BH4), red (BH3), green (BH1) and blue (BH2). (F) FACS assays of Annexin V staining in HT1080 cells. Chimeric GFP constructs encoding GFP alone, or fusions of GFP with full-length Bfl-1 or Bax or with the various membrane-active α-helices corresponding to the C-terminal part of Bfl-1 or Bax, i.e. α5, α6 (PFD, pore forming domain) and α9 (FE, final exon) are represented. The α-helical topology of Bax and Bfl-1 corresponds to the structures solved in aqueous environment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Herman1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Suzuki1" target="_blank">[50]</a>. Transfected cells were stained for phosphatidylserine exposure using Cy3-conjugated Annexin V and the percentage of apoptotic GFP-expressing cells was determined by FACS 24 hours post transfection (right panel). Death of GFP-expressing and staurosporine (STS)-treated cells were also monitored as controls. Graphs shown are representative of three independent experiments.</p

Bfl-1-derived peptides have different abilities to permeabilize the MOM of mitochondria isolated from cultured cells.

<p>Peptides were incubated at different concentrations (10 µM and 25 µM) with isolated mitochondria for the indicated times (5, 15, 30 and 60 min) and the release of cytochrome c was monitored by immunoblot (IB). MitoHsp70 (mHsp70) was used as control indicative of equal-loading and proper isolation of the pellet fraction containing mitochondria (Mito) in comparison to the supernatant fraction (SN). Cytochrome c release assays were performed using iBMKW2 (wild type) and iBMKD3 (double KO Bax/Bak) for all tested peptides. For Bfl-1-α5, wild type MEF and MEF DKO (Bax/Bak −/− double KO) cells were used in parallel.</p

Subcellular localization of the GFP-tagged, Bfl-1/Bax-derived (poly)peptides.

<p>MEF (left panels) and MEF DKO (right panels) cells were co-transfected with mitoDsRed plasmid (encoding DsRed2 fused to the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase) and the GFP-tagged constructs. Subcellular distribution was analyzed by confocal microscopy 24 h after transfection. Confocal images showing GFP (green) and MitoDsRed (red) fluorescence. The DNA staining dye Topro-3 (blue) was used to visualize the nuclei. In merged images, the yellow color shows the co-localization of GFP and MitoDsRed at mitochondria. Scale bar, 10 µm.</p