Effect of hypoxia and/or etoposide on the HIF-1α protein level and HIF-1 DNA binding activity

<p><b>Copyright information:</b></p><p>Taken from "Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines"</p><p>http://www.molecular-cancer.com/content/6/1/61</p><p>Molecular Cancer 2007;6():61-61.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2099441.</p><p></p> A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. , HIF-1α was detected in total cell extracts by western blotting. a-tubulin was used to assess the total amount of proteins loaded on the gel. , after the incubation, nuclear extracts were performed from three independent experiments and hybridized in the ELISA well containing specific DNA probes (TransAM assay). Detection was performed using an anti-HIF-1α antibody. Results are expressed in absorbance (means ± 1 SD, n = 3)

Gene expression profiling, for genes involved in regulating apoptosis, in A549, MCF-7 and HepG2 cells incubated with or without etoposide under normoxic or hypoxic conditions

<p><b>Copyright information:</b></p><p>Taken from "Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines"</p><p>http://www.molecular-cancer.com/content/6/1/61</p><p>Molecular Cancer 2007;6():61-61.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2099441.</p><p></p> Please refer to supplementary data [Additional file ] for results obtained for the 62 genes for which there was a significant variation in expression for at least one of the conditions. Cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours before RNA extraction, reverse-transcription and cDNA hybridization, as described in Materials and Methods. Each value is the average of three ratio values calculated from three independent experiments ± 1 S.D. Mean ratios indicate a fold-increase or decrease in gene expression. Qualitative values are given with + or - signs (according to the inserted table). The red vertical bars correspond to undetected cDNA. Duplicates or unique value are noted with a red 2 or 1 behind the corresponding column

Comparison of the results obtained with real time RT-PCR and DNA microarrays analyses for , , and genes

<p><b>Copyright information:</b></p><p>Taken from "Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines"</p><p>http://www.molecular-cancer.com/content/6/1/61</p><p>Molecular Cancer 2007;6():61-61.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2099441.</p><p></p> After the incubation, total RNA was extracted, submitted to reverse transcription and then to amplification in the presence of SYBR Green and specific primers. was used as the house keeping gene for data normalization. For real-time RT-PCR results, data are given in fold-induction. For DNA microarray results, mean ratios indicate a fold-increase or decrease in gene expression. They are highlighted in blue if statistically non significant, in yellow for quantitative data and in green for qualitative data, given with + or - signs (according to the inserted table)

Gene expression profiling, for genes involved in regulating cell cycle, in A549, MCF-7 and HepG2 cells incubated with or without etoposide under normoxic or hypoxic conditions

<p><b>Copyright information:</b></p><p>Taken from "Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines"</p><p>http://www.molecular-cancer.com/content/6/1/61</p><p>Molecular Cancer 2007;6():61-61.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2099441.</p><p></p> Please refer to supplementary data [Additional file ] for results obtained for the 62 genes for which there was a significant variation in expression for at least one of the conditions. Cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours before RNA extraction, reverse-transcription and cDNA hybridization, as described in Materials and Methods. Each value is the average of three ratio values calculated from three independent experiments ± 1 S.D. Mean ratios indicate a fold-increase or decrease in gene expression. Qualitative values are given with + or - signs (according to the inserted table). The red vertical bars correspond to undetected cDNA. Duplicates or unique value are noted with a red 2 or 1 behind the corresponding column

Global Analysis of Quorum Sensing Targets in the Intracellular Pathogen <i>Brucella melitensis</i> 16 M

Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen <i>Brucella melitensis</i>, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of <i>B. melitensis</i> 16 M. A Δ<i>vjbR</i> mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of <i>Brucella</i>. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the <i>B. melitensis</i> 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of <i>Brucella</i> to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in <i>Brucella</i> and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of <i>Brucella</i> strains to the host environment

Global Analysis of Quorum Sensing Targets in the Intracellular Pathogen <i>Brucella melitensis</i> 16 M

Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen <i>Brucella melitensis</i>, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of <i>B. melitensis</i> 16 M. A Δ<i>vjbR</i> mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of <i>Brucella</i>. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the <i>B. melitensis</i> 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of <i>Brucella</i> to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in <i>Brucella</i> and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of <i>Brucella</i> strains to the host environment

Global Analysis of Quorum Sensing Targets in the Intracellular Pathogen <i>Brucella melitensis</i> 16 M

Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen <i>Brucella melitensis</i>, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of <i>B. melitensis</i> 16 M. A Δ<i>vjbR</i> mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of <i>Brucella</i>. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the <i>B. melitensis</i> 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of <i>Brucella</i> to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in <i>Brucella</i> and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of <i>Brucella</i> strains to the host environment

MS/MS-identification of proteins detected by SERPA from colorectal cancer cells exposed to hypoxia.

<p>Mapping of spots of interest resulting from the comparison described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076508#pone-0076508-g001" target="_blank">Fig.1</a> and list of identified proteins (p<0.001) obtained using lysates of HCT116 colorectal cancer cells.</p

Hypoxia integration in the SERPA strategy.

<p><b>A.</b> Workflow of the SERPA process including 2DE-gel separation of lysates from either hypoxic or normoxic tumor cells, membrane transfer, immunoblotting with the serum from either control or tumor-bearing mice, and detection of spots of interest. <b>B.</b> Typical immunoblotting patterns resulting from the incubation of 2D-resolved lysates of HCT116 cells exposed to normoxia or hypoxia, with the indicated mouse serum. In the bottom panels, proteins of the lysates are labelled with Cy dye (red) and fixed antibodies are detected with an anti-mouse secondary antibody (green spot); arrow indicates the presence of a protein exclusively detected in the lysates of hypoxic tumor cells by antibodies from the serum of tumor-bearing mice.</p

Validation of phospho-eEF2 protein as the target of autoantibodies in mice bearing colorectal HCT116 tumors.

<p><b>A.</b> Representative immunoblotting of 2D-separated lysates of hypoxic HCT116 cells with a commercial antibody against eEF2. Proteins of the lysates are labelled with Cy dye (red) and secondary antibody is conjugated to horseradish peroxidase (green spots). Positive signal is obtained for several spots of the same molecular weight but differing by their pI value. <b>B.</b> Comparison of the eEF2 spots detected with a commercial antibody against total eEF2 (top), the serum from tumor-bearing mice (middle) and a commercial antibody against phospho-Thr56 eEF2 (bottom). Spot 4 (rightmost spot) corresponds to the unphosphorylated form of eEF2 while the other spots correspond to multi-phosphorylated forms of the protein; spot 3 (second spot from the right) corresponds to the preferential monophosphorylated form of eEF2 (on Thr56).</p