Fig 2: Hirudin suppresses TGF-β-induced epithelial-mesenchymal transition via S1P/S1PR2/S1PR3 signaling-mediated PAR1 inhibition. (A) Expression levels of N-cad, Slug and E-cad in HK-2 cells were determined using western blotting. (B and C) α-SMA expression in HK-2 cells was analyzed by immunofluorescence analysis. Scale bar, 100 µm. Error bars represent the mean ± SEM from three independent experiments. ***P<0.001 vs. the control. ###P<0.001 vs. TGF-β. ∆P<0.05 and ∆∆∆P<0.001 vs. TGF-β+hirudun (1 mg/ml). S1PR, sphingosine-1-phosphate receptor; PAR1, protease-activated receptor 1; N-cad, N-cadherin; E-cad, E-cadherin; α-SMA, α-smooth muscle actin; S1P, sphingosine-1-phosphate; TFL, TFLLR-NH2.

Fig 3: Intra-tumor heterogeneity of the coagulome of malignant cells from individual OSCC. Violin plots showing the expression of the core coagulome genes (F3, PLAU, PLAT, PLAUR, F2R and SERPINE1) in cancer cells from different OSCC tumors analyzed in GSE103322. Each individual tumor was identified according to the study by Puram et al. [28]. The red dots represent the median value for the expression of each gene.

Fig 4: Hirudin inhibits TGF-β-induced PAR1 upregulation in HK-2 cells and the renal tissues of UUO mice. Relative (A) mRNA levels and (B) protein expression levels of S1PR1-4 and PAR1 in HK-2 cells were detected by RT-qPCR and western blot analysis, respectively. (C) Expression levels of proteins, including S1PR1-4 and PAR1, in the renal tissues of UUO mice were determined by western blotting. (D) Relative mRNA levels of PAR1 in HK-2 cells were determined by RT-qPCR. PAR1 expression in (E) HK-2 cells and (F) renal tissues of UUO mice was quantified by western blotting. Error bars represent the mean ± SEM from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. the control. #P<0.05, ##P<0.01 and ###P<0.001 vs. TGF-β or UUO. UUO, unilateral ureteral ligation; PAR1, protease-activated receptor 1; S1PR, sphingosine-1-phosphate receptor; RT-q, reverse transcription-quantitative.

Fig 5: Polar plots showing the mRNA expression of the core coagulome genes (F3, PLAU, PLAT, PLAUR, F2R and SERPINE1) in different cell subpopulations of OSCC. The graphs show the relative expression of each gene in the different cell types (malignant cancer cells, T cells, dendritic cells, macrophages, endothelial cells and fibroblasts) using scRNA-seq data from GSE103322. In each case, the cell type with the highest expression (median) was set as maximum. Note that cancer cells are shown in red.