Additional file 4: of Inflammasome and toll-like receptor signaling in human monocytes after successful cardiopulmonary resuscitation

Time-dependent monocyte mRNA expression in 30-day nonsurvivors following cardiac arrest. Shown are monocyte mRNA expression levels of TLR2, TLR4, IRAK3, IRAK4. NLRP1, NLRP3, AIM2, PYCARD, CASP1, and IL-1β in 30-day nonsurvivors in the first 12 h (CPR t1: n = 18), after 24 h (CPR t2: n = 18), and after 48 h (CPR t3: n = 11) following ROSC. Statistical hypothesis testing was performed using the Kruskal–Wallis test and post-hoc analysis with all-pairwise comparison using the Dunn–Bonferroni approach (*p value ≤0.05; **p value ≤0.01; ***p value ≤0.001). (DOCX 42 kb

Blockade of monocyte binding to endothelial cells by APC in different concentrations under flow conditions.

<p>Monocyte binding to HUVECs in dynamic adhesion assays after 5 min (left) and 10 min venous flow (middle), and after 1 min arterial flow (right). Pre-treatment of HUVECs with 10 µg/mL activated protein C (drotrecogin alfa) (dark grey bars) diminished monocyte adhesion compared to 1 µg/mL (light grey bars) and 5 µg/mL (grey bars) drotrecogin alfa. (^##<i>p</i><0.005; ^#<i>p</i><0.05 vs. 1 µg/mL APC).</p

Equal blocking of monocyte binding to endothelial cells by anti-EPCR and APC under flow conditions.

<p>Monocyte binding to HUVECs in dynamic adhesion assay after 5 min (left) and 10 min venous flow (middle), and after 1 min arterial flow (right). Pre-treatment of HUVECs with anti-EPCR (dark grey bars), anti-Mac-1 (light grey bars), or with activated protein C (drotrecogin alfa; crosshatched bars) diminished monocyte adhesion to an equal extent compared to control. (*** <i>p</i><0.0001; ^###<i>p</i><0.0005; ^##<i>p</i><0.005; ^#<i>p</i><0.05 vs. no blocking; ns  =  statistically not signifiant).</p

Specific binding of CHO cells expressing activated Mac-1 to recombinant EPCR in static adhesion assay.

<p>Specific binding of CHO cells transfected with permanently activated Mac-1 (Mac-1+ CHO cells; black bars) to soluble, recombinant EPCR in a static adhesion assay (left). Blocking with an anti-Mac-1 antibody resulted in loss of EPCR binding capacity of Mac-1+ CHO cells (right). Native CHO cells without transfection of Mac-1 served as a negative control (light grey bars). (^###<i>p</i><0.0005; ns  =  statistically not significant vs.native CHO cells; *** <i>p</i><0.0001).</p

Monocytes bind sEPCR in a concentration dependent manner.

<p>Monocytes bind soluble, recombinant EPCR in a concentration dependent manner in flow cytometry analysis. GST tagged-EPCR was detected with an Alexa Flour-labeled anti-GST antibody. Secondary antibody and isotype IgG served as controls (black bars on the left and second from left). (*** <i>p</i><0.0001; # <i>p</i><0.05 vs. controls).</p