Summary of the degree of inhibition obtained with the PI3 kinase inhibitors on CXCL-8 and GMCSF induced neutrophil migration.

<p>% inhibition</p><p>+++ ~100%</p><p>++ >50%</p><p>- Not significant</p><p>ND: Not done</p><p>Summary of the degree of inhibition obtained with the PI3 kinase inhibitors on CXCL-8 and GMCSF induced neutrophil migration.</p

An antagonist of PI3Kα inhibits CXCL8 induced chemotactic migration in a dose and time dependent manner.

<p>a) Neutrophils were pre-treated for 30 mins with varying concentrations (from 0.1μM to 10μM) of the PI3Kα selective inhibitor PIK75, and then stimulated with 100ng/ml CXCL8 in the gradient assay. b) Neutrophils were pre-treated with 1μM of PIK-75 for 30 minutes or 2 hours, prior to construction of the migration gel and the addition of 100ng/ml of CXCL8 in the gradient assay. Results are shown as mean ±SEM; n = 4, except for CXCL8, 1μM and the corresponding DMSO control where n = 11 ***p<0.001, **p<0.01. <b>Antagonists of PI3Kδ and α have additive effects on inhibition of CXCL8 mediated neutrophil chemotaxis.</b> Neutrophils were pre-treated for 30 mins with either 2μM of the PI3Kα selective inhibitor, PIK-75, 2μM of the PI3Kδ selective inhibitor, PIK-274 or 1μM of each and then stimulated with 100ng/ml CXCL8. Results are shown as mean ±SEM (n = 4) *p<0.05. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116250#pone.0116250.g006" target="_blank">Fig. 6c</a>)</p

An antagonist of PI3Kγ inhibits CXCL8 mediated chemokinetic but not chemotactic migration and GM-CSF mediated chemokinetic migration at a sub-optimal concentration of GM-CSF.

<p>Neutrophils were treated with 10μM PI3Kγ selective inhibitor for 30 mins prior to the addition of CXCL8 (100ng/ml, a) or GM-CSF (0.5ng/ml, 50ng/ml, b). Data shown are mean ±SEM (n = 3) except for non gradient CXCL8 where n = 4 and GM-CSF where n = 6 ***p<0.001.</p

An antagonist of PI3Kδ inhibits both chemotactic and chemokinetic migration mediated by CXCL8 and GM-CSF-.

<p>Neutrophils were treated with 1μM and 10μM of the PI3Kδ selective inhibitor PIK-274 for 30 mins prior to the addition of CXCL8 (100ng/ml, a) or 0.5ng/ml GM-CSF (b). Data shown are mean ±SEM (n = 3) except for gradient DMSO and 1μM of PIK-274 where n = 8. *p<0.05, ***p<0.001.</p

Neutrophils express the class I PI3K catalytic subunits α,δ and γ.

<p>RNA was isolated from unstimulated neutrophils. RT-PCR was carried out using primers for each of the four class I PI3-kinase catalytic isoforms and β-actin as a control. n = 1 representative of three experiments. The identity of each of the bands was confirmed by excising the band and sequencing the DNA product.</p

Wortmannin inhibits both CXCL8 and GM-CSF-mediated neutrophil migration.

<p>Neutrophils were pre-treated for 30 mins with 50nM wortmannin, and then stimulated with a) CXCL8 (100ng/ml) or b) GM-CSF (0.5ng/ml, 50ng/ml). Results are shown as mean ±SEM (n = 7) except for DMSO controls and 0.5ng/ml GM-CSF where n = 3, ***p<0.001.</p

Neutrophil migration is dose dependent in both the gradient and non-gradient assays.

<p>a) migration in response to CXCL8, *significantly greater as compared to control cells, p<0.05, n = 3 except for non gradient control and 100ng/ml where n = 5. b) The direction of movement for each neutrophil in the non gradient and gradient assay in response to 100ng/ml of CXCL8 is illustrated in the vector diagrams which show chemokinetic migration in the non-gradient assay and a chemotactic pattern of migration in the gradient assay. c) Neutrophil migration to GM-CSF is dose dependent in both the non gradient and gradients assays. *significantly greater as compared to control cells, p<0.05, n = 3 except for gradient control and 50ng/ml where n = 7 and 6 respectively. d). The direction of movement for each neutrophil in the non gradient and gradient assay in response to 50ng/ml of GM-CSF is illustrated in the vector diagrams, These show that the pattern of migration to GM-CSF was chemokinetic in both the gradient and non-gradient assays.</p

CXCL8 and GM-CSF induce the phosphorylation of Akt.

<p>Neutrophils were treated with wortmannin (50nM) or the PI3K (1M), (10M) and (10μM) selective inhibitors for 2 min in combination with 500ng/ml of CXCL8 (a,b) or 50ng/ml GM-CSF (c,d). Western blots were carried out using the antibodies Phosphorylated Akt and total Akt (a,c). Densitometry was performed on the western blot films (b,d). The western blots shown are an example of 1 of the 3 carried out which all demonstrated the same pattern. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116250#pone.0116250.g007" target="_blank">Fig. 7b</a> CXCL8+DMSO significantly increased phosphorylation of Akt compared to the unstimulated control (p<0.05) and PIK-75 (PI3kinase α) caused significant inhibition of phosphorylation (p<0.05). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116250#pone.0116250.g007" target="_blank">Fig. 7d</a> the PI3kinase δ inhibitor PIK-294 significantly inhibited GM-CSF+DMSO mediated phosphorylation (p<0.05)</p

IC50s of the PI3 kinase isoform inhibitors.

<p>IC50s of the PI3 kinase isoform inhibitors.</p