4 research outputs found

    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

    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

    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
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