APSs conferred elevated expression in HL-60 macrophages.
(A, B and C) Schematic representations of UTR-Nluc reporters with UTR mutations are shown. The AU content in the first 100 bases of the 3’ UTR of these UTR-Nluc reporter mutants are shown in line charts. Positions 1 to 91 of the 3’ UTR of TNF was inserted in between positions 285 and 286 of the CXCL8-3’ reporter to generate the CXCL8-3’::disTNF-3’(1–91) mutant. The expression plasmids for these UTR-Nluc reporter mRNAs were transfected into parallel cell cultures. The resulting Nluc protein and mRNA expression levels were quantified via luciferase assay and real-time PCR, respectively, after overnight incubation. The ratio of UTR-Nluc over Cntrl-Nluc expression was then determined and presented as log2 values. Each graph symbol (squares or triangles) is the result of a replicate experiment. Replicate experiments were performed on different days. (D) The 3’ UTR of TNFAIP6, IFNG and IL2 contain putative APSs. (E) From 14 fractions spanning the entire sucrose gradients displayed in Fig 1C, the levels of specific TNFAIP6 mRNA were quantified via real-time PCR and presented as a percentage of the sum of all fractions. Replicate experiments were performed on different days and the data from 2 replicates are shown.