ATM inhibition increases p38 activation as well as cytokine transcription and production in stimulated neutrophils. (A) Western blot analysis of phosphorylation of ATM, histone H2A.X, and p38 in neutrophil lysates over a 180-minute time course of LPS stimulation with and without 10 µM KU-55933, representative blot of 4 experiments. (B) Relative levels of phospho-p38 quantified by densitometry in ImageJ software. Data are expressed as signal relative to the loading control GAPDH (n = 4). (C) Effect of p38 and ERK inhibitors on neutrophil IL-8 production in the presence of KU-55933 in response to LPS stimulation. IL-8 concentration in supernatants was measured after 18 hours of stimulation (n = 5) (D). Quantitative real-time PCR analysis of relative IL-8 and MIP-1α transcripts in cDNA from neutrophils stimulated with LPS with and without KU-55933. mRNA was isolated from LPS-stimulated neutrophils at the indicated time points, converted to cDNA, and analyzed by qPCR in triplicate (n = 3). Data are expressed as the relative amount of cytokine transcript divided by the relative amount of housekeeping gene β2-microglobulin (n = 3). Relative transcript amounts were calculated using a standard curve made from serial dilutions of a pooled sample. Data were analyzed using StepOnePlus software. (D) Intracellular IL-8 staining of LPS-stimulated neutrophils in the presence of KU-55933. Neutrophils were incubated with Brefeldin A to block secretion and stimulated with LPS with or without ATM inhibitor. At the indicated time points, cells were fixed and stained with an anti-IL8 antibody and analyzed by flow cytometry. At least 10 000 cells were analyzed per sample. Gray fill represents unstimulated neutrophils, the solid line represents LPS-stimulated dimethyl sulfoxide control, and the dashed line represents LPS-stimulated with 10 µM KU-55933. (B-D) Data are presented as the mean ± SEM of compiled experiments. Asterisks indicate significance: *P < .05, **P < .01, ***P < .001 by paired Student t test.