FIGURE 4.

RET negatively regulates the ATF4 transcriptional activity. A, nuclear retention of ATF4 in RET shRNA TT cells. PARP and DNMT1A (DNA methyl transferase) were used as nuclear markers, and vinculin was used as a cytoplasmic marker. TL, total lysates; Cyt, cytoplasmic fraction; Nuc, nuclear fraction. B and C, RET inhibits ATF4-dependent gene activation. Shown are reporter assays with HEK293T cells transfected with the 4xCRE-Luc reporter (B) and the NOXA-Luc reporter (C) along with expression vectors encoding ATF4-WT, RET-WT, mutant RET (RET-C634W, RET-M918T), and RET kinase-dead mutant (RET-KD). D, ChIP assays indicating ATF4 occupancy at the NOXA promoter in TT cells treated with TKI (10 μm vandetanib for 8 h) and RET shRNA TT cells (left); MZCRC1 cells were treated with 10 μm sunitinib for 8 h (right). Data are shown as means ± S.D. (error bars) from two independent experiments. The statistical analysis used was unpaired Student's t test. Asterisks indicate p value, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.