Figure 3.

5′ UTR of PG1 mRNA suppresses its translation

(A) The 5′ UTR sequences of PG1 and OCT4A were analyzed to assess their effects on protein translation. The start codon AUG is shown in upper case (red), whereas the upstream AUG codons are highlighted in yellow. The underlined region indicates the sequence shared by both.

(B) Upper: Diagram of each DNA construct transfected into cells. Lower: Images of EGFP fluorescence in human embryonic kidney (HEK293A), endometrial cancer (HEC50B, Ishikawa), and colorectal cancer (HCT116, DLD-1) cell lines 2 days after transfection. Scale bars, 50 μm.

(C) Luminescence intensity was measured using a dual-luciferase assay. Left: Firefly/Renilla luciferase ratio in HEK293A cells. Right: Relative luminescence values normalized to the no 5′ UTR data (set to 1) for each cell line. n = 3. Mean ± SD. One-way ANOVA. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, and ∗∗∗∗ = p < 0.0001.

(D) Fluorescence intensity values were normalized to RFP fluorescence when plasmid DNA containing EGFP and 3′ UTR sequences with different lengths of the PG1 5′ UTR sequence was transfected into HEK293A cells. The mean and variability of the 16 randomly captured fields are shown. n = 16. Mean ± SD. This experiment was repeated multiple times to confirm that similar results were obtained. Fluorescence images were captured 2 days after transfection. Scale bars, 100 μm.