Human neutrophils express only cell cycle independent CDKs.

<p>Isolated human neutrophils (N) and promyelocytic HL60 cells (H) were assessed for expression of CDK proteins by western blotting (upper panel). Blots were scanned to determine the CDK:β-actin ratio by densitometry (lower panel; black bars  =  HL60, grey bars  =  neutrophils). Data are mean ± s.d. for three separate cell extracts.</p

Flavopiridol inhibits upregulation of Mcl-1 by GM-CSF.

<p>(a). Neutrophils were incubated with medium alone, or medium containing 100 nM flavopiridol in the absence (open bars) or presence of (filled bars) of 50 ng/ml GM-CSF. Live cells were detected by their ability to retain DiOC₆ and data are mean ± s.d. of 3 separate experiments. * indicates p<0.05 for control versus flavopiridol treated cells. (b). The same cells were extracted and measured for Mcl-1 content by western blotting. Blots were probed for β-actin to confirm equal loading.</p

Inhibition of CDK9 but not cell cycle dependent CDKs increases neutrophil apoptosis.

<p>(a) Isolated human neutrophils were incubated with NU6102 (filled triangle), R-roscovitine (filled square), or LGR1406 (filled diamond) at the concentrations shown. Apoptosis was determined after 15h by assessment of DiOC₆ uptake and flow cytometry, with DiOC₆ low cells taken as apoptotic. Data are mean ± s.d. for four separate experiments. * indicates p<0.05 for treatment versus control. (b) Neutrophils were incubated with flavopiridol for 15 h at the concentrations shown and apoptosis determined by assessment of DiOC₆ uptake. Data are mean ± s.d. for three separate experiments. * indicates p<0.05 for control versus flavopiridol treated cells. (c) Images of Giemsa stained neutrophils cultured in the absence (control) or presence of 100 nM flavopiridol for 15 h.</p

CDK9 activity and cyclin T1 expression decrease as neutrophils age in culture.

<p>Isolated human neutrophils were cultured for 9h and (a) CDK9 protein expression and (b) enzymatic activity were determined and compared with freshly isolated cells (0h). For CDK9 activity protein was immunoprecipitated from neutrophils with a monoclonal anti-CDK9 antibody and activity determined by incorporation of [γ ³²P]-ATP into the CTD of RNA polymerase II. The phosphorylated peptide was isolated on an SDS-PAGE gel and excised for scintillation counting. Data are expressed as % of the value for control freshly isolated cells and are mean ± s.d. for three separate experiments. * indicates p<0.05. (c). Isolated neutrophils were cultured for 0 h or 9 h and expression of cyclin T1 determined by western blotting. Blots were probed with an antibody to β-actin to confirm equal protein loading. The blot shown is representative of 4 separate experiments performed. (d) Western blots of CDK9 expression relative to β-actin were quantitated using densitometry.</p

Endotheliopathy is associated with higher levels of cell-free DNA following major trauma: A prospective observational study - Fig 2

<p><b>Association between (a) syndecan-1 and (b) thrombomodulin and cell-free DNA (cfDNA) according to time points.</b><i>P</i>-values are indicated according to Spearman’s rank correlation coefficient. For both concentrations of (a) syndecan-1 and (b) thrombomodulin, there is a significant correlation with concentration of cfDNA at both time points.</p

Biomarker concentrations at both time points compared between patients that received a blood transfusion and those who did not.

<p>Biomarker concentrations at both time points compared between patients that received a blood transfusion and those who did not.</p

Patient characteristics according to patient cohort.

<p>Patient characteristics according to patient cohort.</p

Endotheliopathy is associated with higher levels of cell-free DNA following major trauma: A prospective observational study - Fig 2

<p><b>Association between (a) syndecan-1 and (b) thrombomodulin and cell-free DNA (cfDNA) according to time points.</b><i>P</i>-values are indicated according to Spearman’s rank correlation coefficient. For both concentrations of (a) syndecan-1 and (b) thrombomodulin, there is a significant correlation with concentration of cfDNA at both time points.</p

Patient characteristics according to requirement for transfusion.

<p>Patient characteristics according to requirement for transfusion.</p

Biomarker concentrations at both time points compared between patients that had surgery between time points and those who did not.

<p>Biomarker concentrations at both time points compared between patients that had surgery between time points and those who did not.</p