Primary macrophages display an elevated rate CXCL8 translation that is attenuated by ERK1/2 signaling inhibitors.

(A) CXCL8 protein and mRNA expression levels in primary macrophages, neutrophils and NK cells. Expression was measured at resting state (not treated, NT) or after overnight activation with 100 ng/mL LPS or 10 ng/mL TNF. (B) Polysome profiles of primary macrophages and HL-60 Mac were obtained from a continuous sucrose density gradient (10–50% sucrose). From 14 fractions spanning the entire sucrose gradient, the levels of specific mRNAs (CXCL8, TNFAIP6, ACTB and RPL27) in each fraction from the sucrose gradients were quantified via real-time PCR and presented as a percentage of the sum of all fractions. The data for the HL-60 Mac repeats were as previously shown in Figs 1C and 5E. (C) Graphical representations of the CXCL8-5’+3’ UTR-Nluc and Cntrl-Nluc reporter mRNAs are shown. The expression plasmids for these Nluc reporters were transfected into parallel cell cultures. After overnight incubation, the resulting Nluc protein and mRNA expression levels were quantified via luciferase assay and real-time PCR, respectively. The ratio of UTR-Nluc over Cntrl-Nluc expression was then determined and presented as log2 value. (D and E) Western blots displaying the expression levels of rpS6 S235/236+phos, rpS6 S240/244+phos, total rpS6, 4E-BP1 T37/46+phos, total 4E-BP1, eIF4E S209+phos, total eIF4E and β-Tubulin. The expression levels of these proteins were compared between primary macrophages and NK cells. Comparisons were also made between primary macrophages treated with the solvent control and the ERK1/2 inhibitor, 1 μM AZD6244, The band signal intensities were quantified and used to calculate the normalized band intensity ratios as described in the mathematical expressions. (F) The expression ratio of CXCL8 protein and mRNA expression in primary macrophages and neutrophils upon overnight treatment with ERK1/2 signaling inhibitors (10 μM U0126 or 1 μM AZD6244) relative to solvent control cells. (A to F) Each graph symbol (squares, triangles or circles) is the result of a replicate experiment. Replicate experiments were performed on different days. Cells from different donors were used for each replicate. For the western blots, two replicates are shown. For panels A and F, CXCL8 protein and mRNA levels were determined via ELISA and real-time PCR, respectively. CXCL8 mRNA levels are standardized to RPL27 as the internal control genes and are relative to the control NT samples.