(A) Flt1 mRNA
0 3
NP PE 6
Supplementary Figure 1 Supplementary Figure 1. Expression of Flt1, IL-6, and IL-8 in the placentas. (A-C) Placentas from women in the NP (n=6) and PE (n=12) groups were cultured for 24 h. mRNA expression of Flt1, IL-6, and IL-8 were measured with the use of qPCR. The results are shown as the mean ± SD.
Differences between treatment groups were identified using unpaired t-tests. *P <
0.05.
(B) IL-6 mRNA
0 1
PE NP 2
(C) IL-8 mRNA
0 1
PE NP
*
2Supplementary Figure 2. Effects of S100A9 on cytokine secretion in HUVECs. (A) HUVECs from women in the NP group (n=4) were pre-incubated in the absence of caspase-1 inhibitor for 1 h before S100A9 treatment (1 µg/ml).
sEng levels in supernatants were measured. (B) HUVECs of women in the NP (n=5) group were incubated with recombinant S100A9 (0.05, 0.1, 0.5, 1 µg/ml) for 24 h. LDH levels in supernatants were measured. (C) HUVECs from women in the NP group (n=4) were pre-incubated in the absence of caspase-1 inhibitor for 1 h before S100A9 treatment (1 µg/ml). LDH levels in supernatants were measured. (D) HUVECs of women in the NP (n=6) group were incubated with recombinant IL-1β (1, 5, 10 ng/ml) for 24 h. sEng levels in supernatants were measured. The results are shown as the mean ± SD. Multiple comparisons were made using one-way ANOVA followed by Bonferroni’s multiple comparison test. *P < 0.05.
(D) sEng secretion
pg/ml
300
0 150
1
Control 5 10
IL-1β (ng/ml)
(A) sEng secretion
pg/ml
6000
0 3000
Control Caspase-1 inhibitor (µM)
- 5 10
S100A9
0 Caspase-1
inhibitor (µM)
- 10 20
S100A9
Absorption value 0.6
0 0.3
0.1 0.5 S100A9 (µg/ml)
Control 0.05 1
(B) LDH
0.6
0.3 (C) LDH
Control
*
* *
*
(A) Mean blood pressure
% from PD10
130
100 110 120
90
PD10 PD14 PD17
WT control WT S100A9 i.p.
NLRP3 KO S100A9 i.p.
(B) Ly6G+ neutrophils in placenta
WT control WT S100A9 i.p. NLRP3 KO S100A9 i.p.
WT control WT
NLRP3 KO
% 50 100
0
CD45+Ly6G+ neutrophils
CD19
Ly6G S100A9 i.p.
* *
*
Supplementary Figure 3. Systemic effect of S100A9 during pregnancy in mice. (A) S100A9 (1 µg/100 µl/mouse) or PBS as a control was administered i.p. once daily from GD10 to GD16 in WT or NLRP3-KO mice. Mean blood pressure was measured and shown as a rate increase from GD10. (B) At GD 17, inflammatory cell infiltration in the placenta was analyzed by FACS. The cell population shown in the frame indicates neutrophils (CD45+/Ly6G+/CD19−) in the placenta and the data of the percentage of neutrophils (CD45+/Ly6G+cells) were shown. The results are shown as the mean ± SD. Multiple comparisons were made using one-way ANOVA followed by Bonferroni’s multiple comparison test. *P < 0.05.
