29 research outputs found

    Cdh1 depletion sensitizes cells to ER stress-induced cell death.

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    <p>(A) Empty vector-transfected or Cdh1-KD cells were treated with DMSO or 0.5, 1, or 2 µg/ml of TM for 9 h or 24 h. Total cell lysates were immunoblotted for cleaved PARP and indicated endogenous proteins. (B) Empty vector-transfected or Cdh1-KD cells were treated with DMSO, 0.5 µg/ml, or 1 µg/ml of TM for 9 h or 12 h. Total cell lysates were analyzed as in (A). (C) Empty vector-transfected or Cdh1-KD cells were treated with solvent (mock) or 1–3 mM DTT for 9 h or 12 h. Total cell lysates were immunoblotted as in (A). (D) <i>Left</i>, empty vector-transfected or Cdh1-KD cells were collected at 9, 12, and 24 h after treatment with DMSO or 0.5 µg/ml of TM for DNA content analysis by flow cytometry. Graph shows percentage of sub-G1 population at each time point. <i>Right</i>, empty vector-transfected or Cdh1-KD cells were collected at 9, 12, and 24 h after treatment with solvent (mock) or 1 mM DTT for DNA content analysis by flow cytometry. Graph shows percentage of sub-G1 population at each time point.</p

    Mechanisms supporting APC/C<sup>Cdh1</sup> activity under ER stress conditions.

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    <p>(A) HeLa cells were treated with DMSO, 0.5 µg/ml of TM alone, or 0.5 µg/ml of TM plus 5 µM of MG-132 for 16 h. Immunoprecipitates of endogenous Cdc27 were immunoblotted for endogenous Cdh1. Immunoprecipitation using IgG served as negative control. ** indicates Cdh1-specific top band; *indicates non-specific bottom band. (B) HeLa cells were treated with DMSO or 1 µg/ml of TM for 16 h. CDK2 or CDK1 antibodies were used to immunoprecipitate endogenous CDK2 or CDK1 complexes, respectively. Immunoprecipitates were then used in <i>in vitro</i> kinase assays using histone H1 as substrate. <i>Left</i>, autoradiography of <sup>32</sup>P-histone H1 phosphorylated by CDK2 complexes. Coomassie stains input of histone H1 in the reactions. Intensity of the autoradioactive bands were quantified by Image J, normalized to histone H1 input, and presented as arbitrary units (A.U.). Immunoblot shows comparable levels of endogenous CDK2 and the indicated proteins before and after TM treatment. <i>Right</i>, autoradiography of <sup>32</sup>P-histone H1 phosphorylated by CDK1 complexes, analyzed as indicated for CDK2. (C) <i>Left</i>, total cell lysates from HeLa cells treated with DMSO or 1 µg/ml of TM for 16 h were immunoblotted for the indicated endogenous proteins. <i>Right</i>, quantification of endogenous Emi1 mRNA levels in HeLa cells treated with DMSO or 1 µg/ml of TM for 16 h, measured by SYBR-green qRT-PCR.</p

    APC/C<sup>Cdh1</sup> is activated under ER stress conditions.

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    <p>(A) <i>Left</i>, HeLa cells were treated with DMSO or 2.5 µg/ml of tunicamycin (TM) for 8 h. Total cell lysates were immunoblotted for the indicated endogenous proteins. GRP78 served as a marker of ER stress. <i>Right</i>, HeLa cells were harvested at the indicated times after addition of DMSO or 2.5 µg/ml of TM. Total cell lysates were immunoblotted for the indicated endogenous proteins. HSP90 was used as a loading control. (B) HeLa cells were treated with DMSO or 1 µg/ml of tunicamycin for the indicated times. <i>Immunoblots</i>, total cell lysates were immunoblotted for the indicated endogenous proteins. <i>Graphs</i>, transcript levels as measured by SYBR-green qRT-PCR and protein levels as quantified by LiCOR-Odyssey software on immunoblots are compared for the indicated proteins. All measurements were normalized to the DMSO-0 h sample with relative value of 1. (C) HeLa cells were transfected with pSUPER (empty vector) or pSUPER-Cdh1-shRNA (Cdh1-KD). 24 h after transfection, cells were treated with DMSO, 1 µg/ml of TM alone, or 1 µg/ml of TM plus MG-132 (2 or 5 µM) for 16 h. Total cell extracts were immunoblotted for the indicated endogenous proteins.</p

    Depletion of Cdh1 overcomes ER stress-induced G1 delay.

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    <p>(A) Empty vector-transfected and Cdh1-KD HeLa cells were treated with DMSO or 2.5 µg/ml of TM for 8 h. <i>Left</i>, knockdown efficiency was verified by immunoblotting of endogenous Cdh1. <i>Right</i>, cell cycle distribution of the same cells was analyzed by FACS. Graph quantifies the increase in percentage of cells in G1 after treatment with 2.5 µg/ml of TM for 8 h, normalized to the percentage in DMSO-treated samples. (B) Empty vector-transfected and Cdh1-KD cells were then treated with DMSO or 1 µg/ml of TM for 16 h. <i>Left</i>, knockdown efficiency was verified by immunoblotting of endogenous Cdh1. <i>Right</i>, graph quantifies the increase in percentage of cells in G1 after treatment with 1 µg/ml of TM for 16 h, normalized to the percentage in DMSO-treated samples.</p

    Prolyl hydroxylation of ATF4 on aa 60 and 235 by PHD1/3 limits ATF4 availability.

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    <p>(A–B) Specificity of shRNA used to KD PHD1/3. <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were infected with scrambled control or PHD1 shRNA and PHD3 shRNA alone or in combination, and treated with TM (1 µg/ml) for 6 h. The relative mRNA levels of PHD1 (A) and PHD3 (B) were determined by qPCR. The results are shown as the mean values ± S.E. of three independent experiments. Three independent shRNA were used to confirm the changes shown. (C) PHD1 and PHD3 cooperation is required and mediate the effect of Siah1a/2 on TM-induced CHOP transcription. <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were infected with different PHD1 shRNA or PHD3 shRNA, or their combination, or scrambled shRNA. Cells were treated with TM (1 µg/ml) and collected 6 h later. The relative transcription levels of CHOP were determined by qPCR. (D) PHD1 and PHD3 mediate the effect of Siah1a/2 on hypoxia-induced CHOP transcription. <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were infected with PHD1 shRNA and PHD3 shRNA alone or in combination, or control vector and exposed to 1% O<sub>2</sub> for 6 h. The relative mRNA level of CHOP was determined by qPCR. (E) PHD3 protein is induced in <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells. WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were exposed to 1% O<sub>2</sub> for 24 h prior to the analysis for the expression of HIF-1α, PHD3 and β-actin by Western blotting. The arrow points to the position of the endogenous PHD3 protein. (F) Annotated MS/MS spectra resulting in the identification of proline hydroxylation sites at P60 and P235. Identified fragment ions are shown, as are the detected sites of peptide backbone cleavage; <i>m</i>/<i>z</i>, mass to charge ratio. Note that site determining fragment ions resulted in localization of both sites of proline hydroxylation. (G) Mutations of the two identified proline hydroxylation sites at P235 and P60 to alanine stabilize ATF4 protein. 293T cells were transfected either with Flag-ATF4 or Flag-ATF4 presenting either a mutation at P60, P235, or both. After 24 h from transfection cells were treated overnight with vehicle, DMOG (0.5 mM; upper panel), or MG132 (5 µM; lower panel) followed by cell harvest and immunoblot analysis of ATF4 and β-actin. *** p<0.0005, ** p<0.005, * p<0.05 compared to ad shRNA scr. (A–D) in the same condition (student's t-test). The Western blot experiments were repeated three times and the qPCR results are shown as the mean values ± S.E. of three independent experiments.</p

    List of common significant differential expression genes among KO versus WT, koTG versus wtTG, and koTM versus wtTM (Figure 4b Venn diagram).

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    <p>List of common significant differential expression genes among KO versus WT, koTG versus wtTG, and koTM versus wtTM (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004348#pgen-1004348-g004" target="_blank">Figure 4b</a> Venn diagram).</p

    ATF4 transcriptional activity following oxygen glucose deprivation is Siah1/2 dependent.

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    <p>(A–C) ATF4 transcriptional activity under oxygen and glucose deprivation is Siah2 dependent. WT or <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were subjected to glucose deprivation (GD) oxygen deprivation (1% O<sub>2</sub>; OD) or their combination (OGD) for 12 h followed by cell harvest and RNA analyses. qPCR analysis was performed for ATF3 (A), CHOP (B) and VEGFA (C) using qPCR. (D–E) MEF cells were either infected with scramble shRNA, Siah2 shRNA (D) or Siah1 shRNA (E) and subjected to glucose deprivation and 1% O<sub>2</sub> (OGD) for 12 h followed by cell harvest and RNA analysis using qPCR to determine mRNA levels Siah2, ATF3, and CHOP. (F) WT or <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEF were subjected to glucose deprivation (GD), oxygen deprivation (1% O<sub>2</sub>; OD) or their combination (OGD) for 12 h followed by cell harvest and RNA analysis using qPCR for ATF4 mRNA levels. (G) WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were exposed to glucose deprivation and 1% O<sub>2</sub> (OGD) for 12 h prior to the analysis for the expression of ATF4, PHD1, PHD3 and β-tubulin by Western blotting. Arrow points to the position of the endogenous PHD3 protein. (H) WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were subjected to glucose deprivation and 1% O<sub>2</sub> (OGD) for 12 h prior to the treatment with the protein synthesis inhibitor cycloheximide (40 µg/ml) for 15, 30, and 60 minutes. Cell lysates were subjected to Western blotting using ATF4 and PHD3 antibodies. β-tubulin served as the loading control. Arrow points to the position of the endogenous PHD3 protein. *** p<0.0005, ** p<0.005, * p<0.05 compared to Siah1a/2 WT (A–C, F) or scr. shRNA (D–E) in the same condition (student's t-test). The Western blot experiments were repeated three times and the qPCR results are shown as the mean values ± S.E. of three independent experiments.</p

    Mapping ATF4 and sXBP1 response elements on Siah1a/2 regulatory regions.

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    <p>(A) Overexpression of ATF4 decreases ectopic PHD3 and OGDH protein levels. 293T cells were transfected with myc-tagged PHD3 or flag-tagged OGDH, and 12 h later were infected with ATF4 virus for 24 h. Whole cell lysates were analyzed by Western blotting with the indicated antibodies. Three independent experiments were carried out and Western blot bands corresponding to PHD3 and OGDH were quantified and normalized against β-actin (graph on the right). (B) Overexpression of ATF4 decreases AKAP121 and increases HIF-1α protein level. <i>Atf4<sup>−/−</sup></i> MEFs were infected with ATF4 virus for 24 h and then exposed to hypoxia (1% O<sub>2</sub>) for 8 h. Whole cell lysates were analyzed by Western blotting with the indicated antibodies. (C) Schematic diagram of the human Siah2 promoter, showing the sequence of ATF4 and sXBP1 response elements. The transcription start site is marked as +1. (D) Mapping ATF4 response element on Siah2 promoter. A 2 Kb fragment containing the upstream and the 5′-UTR domains were cloned into luciferase constructs, in which the putative ATF4 binding site (ATF4M; +308 bp) within the 5′-UTR region was mutated. Siah2 promoter activity was monitored in MEFs infected with either GFP or ATF4 expressing adenovirus and 24 h later cells were harvested and proteins were used to perform luciferase assays and to perform Western blotting with the indicated antibodies. (E) ChIP confirms ATF4 occupancy of corresponding Siah2 promoter site. MEFs were exposed to TG for 5 h and ChIP analysis was performed using antibodies to ATF4 or control IgG as indicated. A set of primers was used to quantify a 214 bp fragment containing the ATF4 site in position +308 using qPCR. (F) Mapping the XBP1 response element in the Siah2 promoter. A 2 Kb fragment containing the upstream and the 5′-UTR domains was cloned into luciferase constructs, in which the putative sXBP1 binding site (XBP1M; +287) within the 5′-UTR region was mutated. Siah2 promoter activity was monitored in MEFs infected with adenoviruses expressing either GFP or sXBP1. After 24 h, cells were harvested and proteins were used to perform luciferase assays. (G) ChIP confirms XBP1 occupancy of corresponding Siah2 promoter sites. MEFs were exposed to TG for 5 h and ChIP analysis was performed using antibodies to XBP1 or control IgG as indicated. A set of primers was used to quantify a 214-bp fragment containing the Xbp1 site in position +287 using qPCR. (H) Mapping the ATF4 response element in the Siah1a promoter. ChIP analysis confirms ATF4 occupancy of corresponding Siah1a intron site. MEFs were exposed to TG for 5 h and ChIP analysis was performed using antibodies to ATF4 or control IgG as indicated. A set of primers was used to quantify a 154-bp fragment containing the ATF4 site in position +4,710 using qPCR. *** p<0.0005, ** p<0.005, * p<0.05 compared to ad GFP (D, F) or to control in the same condition (student's t test). The Western blot experiments were repeated three times and the qPCR results are shown as the mean values ± S.E. of three independent experiments.</p
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