Abstract

<p>(A) TM induces Siah2 protein levels. Litter-matched MEF WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells were treated with TM (1 µg/ml) for 10 h. Whole cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies. (B) TM induces Siah1 protein. Litter-matched MEF WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells were treated with TM (1 µg/ml) for 10 h. Whole cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies. Right panel: ER stress does not induce Siah1b mRNA levels. MEFs from <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells were treated with TM (2 µg/ml) for 6 h and the relative expression of Siah1b mRNA was measured by quantitative real time PCR (qPCR). (C) ER stress induces Siah2 mRNA levels. MEFs from WT animals were treated with the indicated concentrations of TM for 6 h and the relative expression of Siah2 mRNA was measured by qPCR. (D) HeLa and Lu1205 cells were treated with the indicated concentrations of TM for 6 h and the relative expression of Siah2 mRNA was measured by qPCR. (E) ER-stress induction of Siah2 mRNA is attenuated in <i>Atf4<sup>−/−</sup></i> MEFs. Littermate-matched MEFs of the indicated genotypes were subjected to treatment with TM (2 µg/ml), hypoxia (H), or both. RNA prepared 6 h later was used for qPCR to quantify Siah2 transcripts, relative to levels of H3.3A mRNA. (F) CHOP mRNA levels are ATF4 but not hypoxia dependent. MEFs from <i>Atf4</i> WT and <i>Atf4<sup>−/−</sup></i> genotypes were subjected to TM or hypoxia or combined treatment and RNA prepared for qPCR analysis of CHOP transcripts. (G) VEGFA mRNA levels are ATF4 dependent under normoxia. Experiment was performed as indicated in panel E, except that qPCR analysis used the VEGFA primers. (H) <i>Atf4</i> WT and <i>Atf4<sup>−/−</sup></i> MEFs were infected with ATF4-expressing adenovirus. Cell lysates were prepared and the level of ATF4 protein was detected in immunoblots with the respective antibody. β-actin served as the loading control. (I) Ectopic ATF4 expression rescues Siah2 mRNA levels in TM-treated <i>Atf4<sup>−/−</sup></i> MEFs. MEFs of the indicated genotypes were infected with control (β-Gal) or with ATF4 adenoviruses for 24 h, followed by 6 h exposure to TM (2 µg/ml), as indicated. Relative expression of Siah2 mRNA was quantified by qPCR. *** p<0.0005, ** p<0.005, * p<0.05 compared to non treated (C–D) or to ATF4 WT in the same condition (student's t test). The Western blot experiments were repeated three times and the qPCR results are shown as the means ± S.E. of three independent experiments.</p

    Similar works

    Full text

    thumbnail-image

    Available Versions