Fig 2: PTPN2 gene therapy decreased diabetes-induced renal fibrosis. A, Representative images of MASSON, and Sirius Red-sensitive collagen staining in renal sections. B, Representative micrographs showing positive Col I (collagen I), Col IV (Collagen IV), Fn (fibronectin), PAI-1 (plasminogen activator inhibitor-1), and TGF-ß (transforming growth factor-ß) and a-SMA (a-smooth muscle actin) immunostaining in glomerular and tubulointerstitium. C, Quantification of fibrosis (% Sirius Red area), Col I, Col IV, Fn, PAI-1, TGF-ß and a-SMA immunostaining in glomerular and tubulointerstitial compartments. D, Representative Western blot analyses of Col I, Col IV, Fn, PAI-1 and TGF-ß expression in renal cortical lysates. E, Quantitative analysis of the results in D. N: normal; DM: diabetes mellitus; IOD: integrated optical density. Data are mean ± SEM of seven to eight animals per group. *P < 0.05 vs. N + Vehicle; # P < 0.05 vs. DM + Vehicle. Original magnification, ×200 in A and B

Fig 3: PTPN2 inhibited HG-induced STAT activation, STAT-dependent genes, and cell proliferation in vitro. MC and MCT were infected with PTPN2-expressing adenovirus or control adenovirus. After 24 h, cells were stimulated for an additional 4 h with LG or HG. A, Representative Western blot analyses for P-STAT1, P-STAT3 and PTPN2 proteins in total cell extracts from MC. B, Quantitative analysis of the results in A. C, Representative Western blot analyses for P-STAT1, P-STAT3 and PTPN2 proteins in total cell extracts from MCT. D, Quantitative analysis of the results in C. E, Representative Western blot analyses for intercellular cell adhesion molecule-1 (ICAM-1), tumour necrosis factor-a (TNF-a), interleukin-6 (IL-6), collagen I (Col I), collagen IV (Col IV), fibronectin (Fn), plasminogen activator inhibitor-1 (PAI-1), and transforming growth factor-ß (TGF-ß) proteins in total cell extracts from MC. F, Western blot analyses of ICAM-1, TNF-a, and IL-6 expression in total cell extracts from MC. G, Western blot analyses of Col I, Col IV, Fn, PAI-1, and TGF-ß expression in total cell extracts from MC. H, Representative Western blot analyses for ICAM-1, TNF-a, IL-6, Col I, Col IV, Fn, PAI-1 and TGF-ß proteins in total cell extracts from MCT. I, Western blot analyses of ICAM-1, TNF-a and IL-6 expression in total cell extracts from MCT. J, Western blot analyses of Col I, Col IV, Fn, PAI-1 and TGF-ß expression in total cell extracts from MCT. K, Monocyte chemotactic protein-1 (MCP-1) concentration in MC supernatants measured by ELISA. L, Cell proliferation assay in cells transfected with PTPN2-expressing adenovirus or control adenovirus after 48 h of incubation in LG or HG. LG: low glucose; HG: high glucose. Data are mean ± SEM of three experiments in duplicate. *P < 0.05 vs. LG + Vehicle; # P < 0.05 vs. HG + Vehicle

Fig 4: TGF-ß1 induces expression of pro-fibrotic molecules. Relative mRNA expression of (a) a-SMA (b) COL6A2 (c) Fibrillin (d) Fibronectin (e) PAI-1 (f) UPR genes XBP1 and CCT4 (g) Protein expression of pro-fibrotic molecules after TGF-ß1 treatment with densitometry analysis using ImageJ (h). (i) Vimentin expression after 10 ng/mL TGF-ß1 treatment and densitometry analysis (j). The error bars represent standard deviation (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by one way ANOVA).

Fig 5: Expression and activity of thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) in vein endothelial cells and a rat model of deep venous thrombosis. (A and B) TAFI (A) and PAI-1 (B) protein expression levels after treatment with rivaroxaban in vein endothelial cells. (C and D) Activity of TAFI (C) and PAI-1 (D) was detected in vein endothelial cells after treatment with rivaroxaban or PBS. (E and F) Plasma concentration levels of TAFI (E) and PAI-1 (F) were decreased in serum in rivaroxaban-treated rat. (G and H) Activity of TAFI (G) and PAI-1 (H) was also decreased in rivaroxaban-treated rat. (I and J) Activity of fibrinolysis (I) and plasma concentration of fibrinolysis (J) in a rat model of deep venous thrombosis. All data are represented as means ± SEM of triplicate samples. One-way ANOVA revealed a significant effect. *P<0.05 and **P<0.01 vs. the control.