Fig. 2.

Arnt-dependent hypoxic induction of the mouse JMJD3 gene. (A, B) Hepa1c1c7 cells and BpRc1 cells were incubated under hypoxia (< 0.5% O₂) for 16 h; (A) Western blot analyses were performed by using the indicated antibodies. The 14-3-3γ protein was used as the loading control; (B) The relative mRNA levels of mouse JMJD3 were analyzed by qRT-PCR. (C) The putative HREs in the mouse JMJD3 gene are identified by using the MatInspector program (www.genomatix.de) (Cartharius, Frech et al., 2005). The bars indicate the positions of the primer sets used for the ChIP analyses. The nucleotide sequences of putative HRE sites (−0.2 Kb, +3.9 Kb, +4.2 Kb) and flanking regions were shown (lower panel). (D) 3T3-L1 cells were incubated under hypoxia (< 0.5% O₂) for 16 h. ChIP analyses were performed with the indicated antibodies and primer sets of mouse JMJD3 and mouse Stra13 genes. Input values were obtained from samples treated in the same way as the experimental method, except that no immunoprecipitation steps were performed. The values on the y-axis are presented as the percentage input of the average and standard deviation of triplicate determinations of qPCR. (E) MEF were incubated under < 0.5% O₂, 3% O₂, and 21% O₂ (normoxia) for 48 h. ChIP analyses (upper panel) and Western blot analyses (lower panel) were performed using the indicated antibodies. N, normoxia; H, hypoxia (< 0.5% O₂). The p-values were obtained using the Student’s t-test and the significance between the groups is indicated (^**p < 0.05, ^*p < 0.1).