FIGURE 6:

The 5′-leader of CDKN1A V4 mRNA directs preferential translation in response to eIF2α-P. N-TERTs were transfected with a CDKN1A V4 luciferase reporter followed by treatment with TG or 100 J/m² UVB, as indicated. Measurements of (A) mRNA and (B) luciferase reporter activity are represented as histograms. (C) Translational control was determined for V4 and V1 variants of CDKN1A or an ATF4 luciferase in response to 6 h of treatment with TG. Firefly luciferase activity was determined using a dual luciferase assay. (D) N-TERTs were transfected with the depicted reporter plasmids encoding the indicated uORFs fused with the firefly luciferase CDS. Luciferase activity was measured after 24 h of transfection. (E) Wild type and the indicated mutant versions of the CDKN1A V4 reporter were transfected into N-TERT cells, followed by treatment with TG for 6 h. Firefly luciferase activity was measured using the dual luciferase assay. Relative amounts of luciferase reporter mRNA as measured by qPCR are shown to the left in D and E. The red “x” indicates mutations of the putative indication codons. * indicates p < 0.05 and # indicates p < 0.05 compared with the untreated reporter. Error bars represent mean ± SD of three separate experiments.