with the inhibitors Wortmannin, SB203580, MG 101 or GF109203X, respectively (n=4, each). (f,g) Effect of LL-37 on platelet CD40L surface expression after (f) ...
Supplementary Figure 5
5
120
4
90
Count
SSC (log)
a
3 2
CD62P expr.
60 30
1 1
2 3 4 FSC (log)
5
0
2 3 4 5 GP1b-APC (log)
2 3 4 5 CD62P-FITC (log)
b PLA (%)
SSC (log)
5 4 3 2 1 1
2
3 4 FSC (log)
5
1
2 3 4 5 Ly6G-PE (log)
1
Count
120
2 3 4 5 GP1b-APC (log) CD11b expr.
90 60 30 0
2 3 4 5 CD11b-FITC (log)
c
SSC (x103)
PLA (%)
Ly6G+ cells
150 100 50 0
0
100 200 FSC (x103)
1
2 3 4 CD45-APC-Cy7 (log)
1 2 3 4 5 Ly6G-PerCP-Cy5.5 (log)
1
2 3 4 5 CD41-PE (log)
Supplementary Figure 5: Flow cytometry gating strategy (a) Gating strategy was used to measure expression of surface molecules in human or mouse platelets (Fig. 3a,c-i, Fig. 4a-c,e-k, Fig. 7o-r and Suppl. Fig. 7; exemplary demonstrated for P-Selectin surface expression) and to assess binding of fluorescently labeled cathelicidins to human or mouse platelets (Fig. 1c). (b) Gating strategy was used to measure human or mouse platelet-neutrophil aggregates (PLA; Fig. 5a,b,i,j, Fig. 6c, Fig. 7m) and to measure neutrophil activation (Fig. 5c-e; exemplary demonstrated for surface expression of CD11b). (c) Gating strategy was used to assess alveolar and interstitial (including pulmonary vasculature) neutrophil infiltration (Fig. 7d,e,g,h) and platelet-neutrophil aggregates in the pulmonary vasculature (Fig. 7n) in acute lung injury.
Supplementary Figure 6 a
Pltl. Spreading
Vehicle
LL-37 (5 µmol/L)
Thrombin (1 U/mL)
Phalloidin
0
% of max. aggregation
% of max. aggregation
b LL-37 5 µmol/L
25 50
ADP 5 µmol/L
75 100
0
1 2 3 4 Time (minutes)
5
100 80 60 40 20 0
LL-37 ADP 5 µM
c
100
Citrated PRP
80 60 40 20 0
ADP ADP TRAP Coll. low high
% of max. aggregation
% of max. aggregation
Vehicle LL-37 5 µmol/L 80
Heparinized PRP
60 40 20 0
ADP low
ADP high
Coll.
Supplementary Figure 6: Platelet spreading and aggregation (a-c) Platelet spreading and aggregation. (a) Platelet spreading upon incubation of washed human platelets with LL-37. Visualizing of the actin-cytoskeleton with Alexa546-phalloidin (red). Thrombin was used as positive control (images are representative of 3 independent experiments; bar, 10 µm). (b) Aggregation of human platelets in platelet-rich plasma (PRP) upon addition of LL-37 (n=3). ADP served as positive control. (c) ADP-, TRAP (thrombin receptor activator) or collagen-induced platelet aggregation after preincubation with LL-37 (5 µmol/L) in citrate (n=6-8; left) or heparinized (n=5-6; right) human PRP. Graphs show mean and SEM.
