PMC

The SNAT2 AARE-independent, KRB-responsive element is in intron 1. HepG2 cells were transfected with the indicated SNAT2 genomic fragments driving transcription for the Firefly luciferase reporter gene. Two different −512/+770 genomic fragments were tested for the ability to drive transcription, one containing the wild type AARE (WT, nt −709/−717) and one that contains a AARE mutated as described in the Sect. “Materials and methods.” Sixteen hours after transfection the cells were treated with complete MEM medium, MEM lacking histidine (MEM-His), or KRB for 10 h. The control (MEM) value for the SNAT2 −512/+770 fragment WT was set to 1. Statistically significant differences, relative to the corresponding MEM control for each fragment, are indicated by asterisks (P <0.005)

Activation of the SNAT2 AARE by single or total amino acid deprivation. Panel a HepG2 cells were transfected with a SNAT2 genomic fragment (nt −512/+770) containing either a wild type (WT) or a mutated (Mut) AARE driving the Firefly luciferase reporter gene, as described in Sect. “Materials and methods.” The cells were incubated in the amino acid deficient media for 10 h. The data are presented relative to control (WT construct, MEM value), which was set to 1. Statistically significant differences, relative to the MEM values, are indicated by asterisks (P <0.005). Panel b HepG2 cells were transfected as described for Panel a, and then the cells were incubated for 10 h in either complete MEM, MEM lacking histidine (MEM-His), amino acid-free Krebs–Ringer bicarbonate buffer (KRB) or amino acid-free Earle’s balanced salt solution (EBSS). The MEM value of the luciferase activity for the wild type control was set to 1. For each treatment, six replicates were performed and statistically significant differences between the WT and the mutated AARE constructs are indicated by asterisks (P <0.005)

Activation of the amino acid responsive pathway by limitation of individual amino acids. HepG2 hepatoma cells were incubated for 2 h in either amino acid complete MEM or MEM lacking the indicated amino acid. Whole cell protein extracts were subjected to immunoblotting for p-eIF2α, total eIF2α, ATF4, actin, total 4E-BP1, or p-4E-BP1, as described in the Sect. “Materials and methods.” The degree of eIF2α phosphorylation is depicted relative to total eIF2 protein present and the data normalized to the value obtained for the amino acid complete MEM (Panel a). The ATF4 content is normalized to actin (Panel b). The abundance of ATF4 in the amino acid-complete MEM was below detection, so the results are presented as the relative values. The data depicted in Panels a and b are the average ± standard deviations for three or more independent experiments (except for Phe which is the average of two samples), and a representative blot is shown in the inset. The abundance of total 4E-BP1 and p-4E-BP1 is shown in Panel c. Total amino acid starvation (−AA) was achieved by incubation in EBSS