Figure 4: ATF4 activates GPT2 transcription in PIK3CA mutant cells.

(a,b) ATF4 protein levels correlate with that of GPT2 in PIK3CA WT and mutant clones. Western blot of lysates of cultured cells (a) or lysates of the xenograft tumours formed by the HCT116 clones (b). (c) Overexpression of ATF4 in HCT116 PIK3CA WT increases GPT2 protein levels. HCT116 PIK3CA WT cells were infected an adenovirus expressing ATF4 (ad-ATF4). (d) Knockdown of ATF4 in the HCT116 PIK3CA mutant clone decreases GPT2 mRNA and protein levels. (e) Knockdown of ATF4 reduces proliferation of HCT116 PIK3CA mutant cells. Scrambled (control) siRNA and two independent siRNAs were transfected into HCT116 PIK3CA mutant clone. (f) Knockdown of ATF4 in the HCT116 PIK3CA mutant clone renders the cells less sensitive to glutamine deprivation. (g) Overexpression of ATF4 S219A stabilization mutant in the HCT116 PIK3CA WT clone increases GPT2 protein levels. A FLAG-tagged ATF4 S219A construct was transfected in HCT116 PIK3CA WT cells and stable pools were selected. Cell lysates were blotted with the indicated antibodies. (h) Overexpression of stabilized ATF4 S219A mutant in the HCT116 PIK3CA WT cells increases cell proliferation. (i) Overexpression of stabilized ATF4 S219A mutant in the HCT116 PIK3CA WT clone renders it more sensitive to glutamine deprivation. Control and ATF4 S219A overexpression cells were grown with or without glutamine for 72 h. Apoptotic cells were quantified. (j) Knockdown of ATF4 in a HCT116 PIK3CA mutant clone decreases activity of a GPT2 promoter transcriptional reporter. GPT2 luciferase reporter plasmid was co-transfected with the indicated siRNA and a plasmid expressing β-galactosidase as internal control. Luciferase activities were assayed 48 h post transfection. (k) Sequence of two putative ATF4-binding sites in the GPT2 promoter, which were predicted by TFseach v1.3 and mutant sequences that abolish ATF4 binding. (l) Mutation of ATF4-binding sites reduces GPT2 promoter reporter expression. Empty vector or vector expressing ATF4 were co-transfected with the indicated reporter plasmids in HCT116 PIK3CA WT cells. Data are presented as mean±s.e.m. of three independent experiments. *P<0.05; **P<0.01; ***P<0.001.