The inhibition of ERK1/2 signaling reduced CXCL8 UTR reporter expression in HL-60 macrophages.
(A) Graphical representations of the plasmid-derived UTR-Nluc reporter mRNAs are shown. These plasmids were transfected into parallel cell cultures. Treatments with DMSO solvent control or ERK1/2 signaling inhibitors (10 μM U0126 or 1 μM AZD6244) were performed three hours after UTR-Nluc reporter transfection. 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 circles) is the result of a replicate experiment. Replicate experiments were performed on a different days. (B) Polysome profiles of HL-60 Mac after treatment with 10 μM U0126 or 1 μM AZD6244 for 8 hours. The fractionation was performed on a continuous sucrose density gradient (10–50% sucrose). From 14 fractions spanning the entire sucrose gradient, the levels of specific mRNAs (CXCL8, TNFAIP6 and ACTB) were quantified via real-time PCR and presented as a percentage of the sum of all fractions. The data for the HL-60 Mac controls were previously shown in Fig 1C. Replicate experiments were performed on different days and the data from 2 replicates are shown.