Fig 2: PTPN2 gene therapy protected from diabetes‐associated renal injury in ApoE‐/‐ mice. A, Serum creatinine levels in ApoE‐/‐ mice with established nephropathy after PTPN2 gene therapy. B, Serum BUN levels in ApoE‐/‐ mice with established nephropathy after PTPN2 gene therapy. C, Proteinuria levels in ApoE‐/‐ mice with established nephropathy after PTPN2 gene therapy. D, Representative images of HE and PAS staining in renal sections. E, Glomerular area quantification and PAS + mesangial area analysis in the experimental groups. N: normal; DM: diabetes mellitus. 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 D

Fig 3: PTPN2 gene therapy inhibited STAT activation in vivo. A, Representative micrographs showing positive PTPN2, P‐STAT1 and P‐STAT3 immunostaining in glomerular and tubulointerstitium. B, Quantification of PTPN2 immunostaining in glomerular and tubulointerstitium. C, Quantification of P‐STAT1 immunostaining in glomerular and tubulointerstitium. D, Quantification of P‐STAT3 immunostaining in glomerular and tubulointerstitium. E, Representative Western blot analyses for PTPN2, P‐STAT1, and P‐STAT3 in renal cortical lysates. 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

Fig 4: 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‐α (TNF‐α), interleukin‐6 (IL‐6), collagen I (Col I), collagen IV (Col IV), fibronectin (Fn), plasminogen activator inhibitor‐1 (PAI‐1), and transforming growth factor‐β (TGF‐β) proteins in total cell extracts from MC. F, Western blot analyses of ICAM‐1, TNF‐α, and IL‐6 expression in total cell extracts from MC. G, Western blot analyses of Col I, Col IV, Fn, PAI‐1, and TGF‐β expression in total cell extracts from MC. H, Representative Western blot analyses for ICAM‐1, TNF‐α, IL‐6, Col I, Col IV, Fn, PAI‐1 and TGF‐β proteins in total cell extracts from MCT. I, Western blot analyses of ICAM‐1, TNF‐α and IL‐6 expression in total cell extracts from MCT. J, Western blot analyses of Col I, Col IV, Fn, PAI‐1 and TGF‐β 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 5: HG caused PTPN2 down‐regulation and STAT activation in cultured renal cells. A, Immunofluorescence microscopy of MC (murine mesangial cells) showed PTPN2 staining in green and nuclear staining in blue. Cells were either left untreated or incubated with high glucose (HG, 30 mmol/L Dglucose) for 24 h. B, Immunofluorescence microscopy of MCT (murine tubuloepithelial cells) showed PTPN2 staining in green and nuclear staining in blue. Cells were either left untreated or incubated with HG for 24 h. C, Representative Western blot analyses showing time course of P‐STAT1, P‐STAT3, and PTPN2 induction by low glucose (LG, 5.5 mmol/L D‐glucose) in MC. D, Quantitative analysis of the results in C. E, Representative Western blot analyses showing time course of P‐STAT1, P‐STAT3, and PTPN2 induction by LG in MCT. F, Quantitative analysis of the results in E. G, Representative Western blot analyses showing time course of P‐STAT1, P‐STAT3 and PTPN2 induction by HG in MC. H, Quantitative analysis of the results in G. I, Representative Western blot analyses showing time course of P‐STAT1, P‐STAT3, and PTPN2 induction by HG in MCT. J, Quantitative analysis of the results in I. LG: low glucose; HG: high glucose. Data are mean ± SEM of three experiments in duplicate. *P < 0.05 vs. Control (0 h)