Abstract

<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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