Fig 2: Box plots illustrate the ELISA validation results of four candidate CAL biomarker proteins. Following iTRAQ assay, four candidate serum proteins were validated with ELISA on all serum samples. With statistical significance, CAT and S100A4 kept higher levels in CAL-KD and CAL+KD samples, respectively. CAT—catalase; CLC—galectin 10; FOLR3—Folate receptor gamma; S100A4—S100 calcium-binding protein A4; CAL—coronary artery lesion; KD—Kawasaki disease.

Fig 3: Trans-well assay showed that S100A4 promoted the migration of HCAECs. We used a trans-well kit to examine whether S100A4 affected the migration ability of HCAECs. (a) With the treatment of different S100A4 dosages, 45 pg/mL (20% of the average concentration on CAL+KD subjects), 90 pg/mL (40%), 180 pg/mL (80%), and 225 pg/mL (100%), HCAECs were subjected to a migration ability examination with a trans-well kit. (b) The HCAECs penetrating the insert membrane were quantified by staining and counting (three rounds * eight replications). (c) The cells were quantified by staining and measuring the OD values (three rounds * two replications). Data were presented as mean ± SD. *, ** and *** with a denoted p-value < 0.05, 0.01, and 0.001, respectively. S100A4—S100 calcium-binding protein A4; HCAECs—human coronary artery endothelial cells; CAL—coronary artery lesion; KD—Kawasaki disease; OD—optical density.

Fig 4: LTEM assay showed that the endothelial layer was more susceptible to in vitro neutrophil infiltration with S100A4 treatment. (a) To examine whether the weakened endothelial layer was more susceptible to neutrophil infiltration, we conducted the LTEM assay. (b,c) S100A4 treatment on the endothelial layer allowed more HL-60 cells to penetrate (2700 vs. 1682). (d) By three independent assays (three rounds * replications, one replication failed), more HL-60 cells penetrated the endothelial layer and a 1.5-fold higher LTEM ability was observed with S100A4 treatment. Each dot denoted the relative LTEM ability of one assay. Data were presented as mean ± SD. **with a denoted p-value < 0.01. LTEM—leukocyte trans-well endothelial migration; S100A4—S100 calcium-binding protein A4; HL-60—a neutrophil-like cell line.

Fig 5: Trans-well assay showed that S100A4 promoted the permeability of the endothelial layer composed of HCAECs. We also used trans-well kits to examine whether S100A4 affected the permeability of the endothelial layer. (a) The permeability of the endothelial layer was quantified by counting how much time was needed for trypan blue to reach the lower chamber (n = 3). (b) The permeability of the endothelial layer was quantified by measuring the concentration of total proteins in the lower chamber (n = 3). Data were presented as mean ± SD. *, **, *** and **** with a denoted p-value < 0.05, 0.01, 0.001, and 0.0001, respectively. S100A4—S100 calcium-binding protein A4; HCAECs—human coronary artery endothelial cells.