The elevated expression of <i>CXCL8</i> in macrophages is mediated by its 3’ UTR.

(A)CXCL8 expression in cells transfected with the pcDNA control, CXCL8-full or CXCL8-CDS plasmids. CXCL8-full expresses the full CXCL8 transcript while CXCL8-CDS expresses a mutant with the 5’ and 3’ UTRs deleted. CXCL8 protein levels were determined via ELISA. CXCL8 mRNA levels were determined via real-time PCR (with primers that prime within the coding sequences) and presented as the ratio of CXCL8 divided by the internal control gene, RPL27. (B) Cells transfected with the control plasmid were assumed to only express endogenous CXCL8. The CXCL8 expression observed for these control cells were subtracted from CXCL8-full and CXCL8-CDS to obtain the expression level of plasmid-derived CXCL8, as represented by [CXCL8-full] and [CXCL8-CDS]. These values were used to calculate the “CXCL8 fold change” in expression based on the mathematical expression shown. (C, D, E and F) Schematic representations of the coding sequence (CDS) of secreted Nanoluc (Nluc) fused to the 5’ and 3’ UTRs of CXCL8, TNF, IL6 and TIMP1.The expression plasmids for these UTR-Nluc reporters 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 the log2 fold change in Nluc expression. Nluc mRNA levels were standardized to the NeoR gene expressed from the pcDNA3.1(+), which is the expression plasmid for the UTR- and Cntrl-Nluc constructs. Cells were treated with 100 ng/mL LPS or 10 ng/mL TNF, 3 h after transfection in panel E. The amount of UTR- and Cntrl-Nluc plasmid transfected was halved and replaced with empty pcDNA3.1 plasmid in panel E. (G) Mutant variants of the CXCL8-5’+3’ UTR-Nluc reporter and Cntrl-Nluc mRNAs, with deletions in the secretory peptide signals resulting in cytoplasmic expression of Nluc. (A to G) Each graph symbol (squares, triangles, inverted-triangles or circles) is the result of a replicate experiment. Replicate experiments were performed on different days.