NPM1 expression modulates chemokine gene expression on LPS exposure in human macrophages. Macrophages were obtained by M-CSF treatment of human peripheral blood monocytes for 4 days before transfection and 24 hours after transfection, were exposed to LPS for the indicated times. mRNA was collected for reverse transcription–quantitative PCR analyses of indicated chemokines. (A) Overexpression of a noncleavable mutant of NPM1 (D213A; NPM-MUT) in human cells. ■ indicates control vector; □, NPM-MUT–expressing vector. (Inset) NPM protein level in transfected cells. (B) Down-regulation of NPM1 with siRNA. Reverse transcription–quantitative PCR analyses were performed 3 hours after LPS treatment. Each panel is representative of at least 3 independent experiments. Scr indicates scrambled; and si1 and si2, two different siRNAs targeting NPM1. (C) The ratio between cytokine quantities detected in control and NPM-MUT–transfected macrophage supernatants with or without LPS exposure was quantified and normalized to control pcDNA-transfected cells. (D) Culture supernatant cytokine array analysis of macrophages treated with LPS for indicated times. AU indicates arbitrary units. (E) IL-8 and MCP1 chemokines were quantified with FlowCytomix in the supernatants of control and NPM-MUT–transfected macrophages treated or not treated with LPS and normalized to control pcDNA-transfected cells (3 independent experiments). Mean ± SD of triplicates; **P < .025; ***P < .01; ****P < .001.