Skip to main content
. 2022 Apr 10;19(2):457–473. doi: 10.1080/15548627.2022.2059744

Figure 6.

Figure 6.

Non-canonical uORFs regulate ATG4B and ATG12 translation in human. (A) Schematic representation of WT and mutant human ATG12 or ATG4B 5'UTR-LUC constructs. ATG12 or ATG4B 5’ UTR-containing uORFs (yellow box) with non-AUG start codons (purple triangle) were cloned upstream of the luciferase CDS in WT constructs. The start codons of the uORFs were changed to AAG (green triangle) in the mutant constructs. (B) Relative luciferase mRNA levels were measured in 293 FT cells transfected with WT and uORF mutant ATG4B 5'UTR-LUC constructs under control (nutrient-rich medium) and 4-h HBSS starvation conditions. Mean±SD of n = 3 independent experiments are shown as indicated. ANOVA; ns: not significant. (C) Relative luciferase activities were measured in 293 FT cells transfected with WT and uORF mutant ATG4B 5'UTR-LUC constructs under control (nutrient-rich medium) and 4-h HBSS starvation conditions. Mean±SD of n = 3 independent experiments are shown as indicated. ANOVA; **: p < 0.01, ***: p < 0.005. (D) Relative luciferase mRNA levels were measured in 293 FT cells transfected with WT and uORF mutant (MUT 1 to MUT 4) ATG12 5'UTR-LUC constructs as in (B). Mean±SD of n = 3 independent experiments are shown as indicated. ANOVA; ns: not significant. (E) Relative luciferase activities were measured in 293 FT cells transfected with WT and uORF mutant (MUT 1 to MUT 4) ATG4B 5'UTR-LUC constructs as in (C). Mean±SD of n = 3 independent experiments are shown as indicated. ANOVA; **: p < 0.01, ***: p < 0.005. WT, wild type; HBSS, Hanks’ balanced salt solution.