Abstract

<p>(A) Various forms of stress increase Siah2 mRNA levels. MEFs were treated with H<sub>2</sub>O<sub>2</sub> (50 µM), TM (1 µg/ml), TG (1 µM) or histidinol (2 µM) for 8 h and Siah2 mRNA was analyzed by qPCR. Relative expression shown represents Siah2 transcript level. (B–E) CHO cells were treated with doxycycline (100 ng/ml) (83) and cells were collected after 0, 6, 24, and 96 h. The relative mRNA level of Factor VII (B), CHOP (C), Siah1 (D) and Siah2 (E) were determined by qPCR. The results are shown as the means ± S.E. of three independent experiments. (F) MEF cells were treated with increasing amount of TM and after 3 h the relative mRNA levels of Siah1 and Siah2 were determined by qPCR. (G) Siah2 is induced upon oxygen and glucose deprivation. WT or <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were subjected to glucose derivation (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 levels of Siah2 transcripts. (H–I) ER-stress induction of Siah2 protein and mRNA levels is attenuated in eIF2α AA MEFs. Littermate-matched MEFs of the indicated genotypes were subjected to treatment with TM (1 µg/ml) or TG (1 µM). Cell lysates were prepared 8 h later and levels of ATF4, Siah2 and CHOP proteins were detected by immunoblots. β-actin served as the loading control (H). RNA was prepared 6 h after treatment with TM or TG and was used for qPCR to quantify the levels of Siah2 transcript, relative to levels of H3.3A mRNA (I). *** p<0.0005, ** p<0.005, * p<0.05 compared to control (panels A–G) * p<0.08 compared to WT (panels H–I) (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 at least three independent experiments.</p

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