Abstract

<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

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