Figure 4.

(A) Schematic representation of the human 5’UTR ATF4 Luciferase reporters (WT- and mut-ATF4 FLuc) consisting of the human WT- and A235G (mut)- ATF4 5′-UTR fused to Firefly luciferase (FLuc) gene. (B) Hep3B cells are co-transfected with either Fluc-expressing vectors or the control plasmid expressing Renilla luciferase (RLuc). Cells are then treated with Thapsigargin (thap) to induce translation of FLuc, the activity of which is measured in the cell extracts and expressed relative to RLuc. The relative values of firefly luciferase were shown as the average of three biological replicates. Error bars correspond to the S.D. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (C) Hep3B stably expressing either a control shRNA (shNT; left panels), shALKBH5 (middle panels), or shFTO (right panels) are co-transfected with a luciferase-expressing vector containing either the WT- or A235G (mut)- ATF4 5′-UTR fused to FLuc gene, and the control plasmid expressing RLuc. Cells are then treated with Thap to induce translation of FLuc, the activity of which is measured. The relative values of firefly luciferase were shown as the average of three biological replicates. Error bars correspond to the S.D. **** p ≤ 0.0001, ns: not significant. Original images can be found in Supplementary Materials.