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. 2013 Jun 25;41(16):7683–7699. doi: 10.1093/nar/gkt563

Figure 2.

Figure 2.

Role of GCN2 and ATF4 in the AARE-regulated p62 transcription in response to amino acid starvation. (A) Effect of leucine limitation on p62 transcription activity. The measurement of p62 heterogeneous nuclear RNA was determined using primers spanning the intron1 as described in Materials and Methods. The graphs show means ±S.E.M. of three independent experiments. (B) Role of GCN2 and ATF4 in the amino acid regulation of p62 transcription. Wild-type, GCN2 −/− and ATF4 −/− MEFs were incubated either in control (+leu) or leucine-free medium (−leu) and harvested after 6 h, and total RNA was analyzed for p62 mRNA content. The graphs show means ± S.E.M. of three independent experiments. t tests have been performed to compare the means. The asterisks indicate a P value of ≤0.05 relative to the +leu medium value. (C) Role of GCN2 and ATF4 in maintaining the p62 protein content in amino acid-starved cells. Immunoblot analysis of p62, phosphorylation of eIF2α, ATF4 and eIF2α protein content were performed from MEFs incubated for 6 h either in control (+leu) or leucine-free medium (−leu). Signal intensities of p62 (three experiments per group) were quantified using Image J software. The asterisks indicate a P value of ≤0.05 relative to the +leu medium value. (D) Comparison of the three p62 sequences (seq1: −1345/−1360, seq2: −934/−949, seq3: +10 053/+10 038) with the Trb3 AARE (+287/+272, +320/+305, +338/+353), the Chop AARE (−295/−313), the Atf3 AARE (−27/−12), the Asns AARE (−72/−57) and the Snat2 AARE (+724/+709). The position of the minimum AARE core sequence is indicated by the gray box. The resulting minimum consensus sequence is shown at the bottom (M = A or C; H = A or C or T). (E) Identification of an AARE in the p62 promoter. MEFs were transiently transfected with LUC constructs containing deletions of the mouse p62 promoter or containing a p62 genomic fragment (−1485 to −1235) with wild-type (WT-AARE: 5′-TGATGACAC-3′) or mutated (Mut-AARE: 5′-CTAGTACAC-3′) AARE (−1360 to −1345) inserted 5′ to the TK promoter. Twenty-four hours after transfection, cells were incubated for 16 h in control DMEM F12 (+leu) or in DMEM F12 lacking leucine (−leu) and assayed for LUC activity. The three putative AARE sequences are indicated by the gray box. The graphs show means ± S.E.M. of three independent experiments. t tests have been performed to compare the means. The asterisks indicate a P value of ≤ 0.05 relative to the +leu medium value.