SFN pre-incubation increases the GSH/GSSG ratio in primary neutrophils.

<p>GSH (A) and GGSG (B) concentrations, along with the GSH/GSSG ratio (C) were measured using the DTNB recycling assay and expressed relative to protein content. Significant differences were calculated with Tukey’s multiple-comparison test where*P<0.05, and ***P<0.001.</p

SFN pre-incubation decreases neutrophil respiratory burst.

<p>Peak relative light unit (RLU) values for neutrophils stimulated with (A) 1µM fMLP, (B) opsonised <i>S. aureus</i> (300 bacteria/neutrophil) (C) <i>F. nucleatum</i> bacteria x100/neutrophil and (D) 10nM PMA in the presence or absence of SFN treatment were plotted and significant differences were calculated using Tukey’s multiple-comparison test, where, * p<0.05 and **p<0.01.</p

A possible mechanism for the up-regulation of NADPH oxidase activity under redox stress.

<p>The pathway leading to the formation of GSH by the action of γ-glutamylcysteine synthetase (γGCS) is blocked by buthionine sulfoximine (BSO), inducing artificial stress condition in dHL60 cells. Decreased cellular GSH/GSSG ratio may activate redox sensitive enzymes such as ASMases and nuclear translocation of Nrf2. ASMases may favour lipid raft formation and thereby clustering active NADPH oxidase complexes in the outer membrane, subsequently liberating extracellular O₂^.-.</p

Effect of SFN on Nrf2 protein expression and activity in primary neutrophils.

<p>(A) Western blotting (left) and quantification (right) of total Nrf2 expression in the presence or absence of SFN treatment. (B) Western blotting (left) and quantification (right) of expression of Keap-1, Values are shown normalised to β-actin. (C) Analysis of Nrf2-DNA binding activity by trans-AM nuclear activity assay. A representative experiment of the three is shown. *** P< 0.01. (D) qPCR analysis of GCLC and GCLM gene expression levels of neutrophil mRNA from patients compared to control. (E) qPCR analysis of GCLC, HMOX1, GCLM and NQO1 gene expression levels in neutrophils treated with 5 µM SFN for 16 hours, values normalised to actin.</p

Effects of BSO and SFN pre-incubation on dHL60 cell GSH/GSSG ratio and respiratory burst.

<p>dHL60 cells were treated with 10µM BSO or 5µM SFN for 16 hours. GSH (A) and GSSG (B) concentrations were measured by the DTNB recycling assay in order to determine the GSH/GSSG ratio (C). Mean peak lucigenin chemiluminescence generated by dHL60 cells was plotted (RLU ± standard error of the mean) (D). Data represent three independent experiments of three replicates. Significant differences were calculated with one way ANOVA followed by Tukey’s multiple comparison test, where *P<0.05.</p

Effects of oxidative stress on ASMase activity and LR rearrangement.

<p>(A) BSO (10µM) pre-treated and untreated dHL60 cells were treated with or without desipramine (10 µM, 1 hour) and 5µM SFN. Cells (1×10⁷) were collected on ice-cold PBS, pelleted and lysed before analysing ASMase activity. (B) LRs from dHL60 cells were extracted with MNE buffer containing 1% triton X-100 and applied to discontinuous sucrose gradients, as described in Methods. Proteins were extracted and immunoblotted using anti-flotillin-1 as the raft protein detecting antibody. (C) Quantification of flotillin in lipid raft fractions 3 and 4 compared to total expression.</p