Fig 1: PARP1 Mediated High Glucose-induced CpG Island Methylation in TFPI2 Promoter. (A) The CpG islands in the promoter of the TFPI2 region immediately upstream -372 bp to -57 bp, which are relative to the translation start sites, were analyzed, wherein a total of 10 CpG islands were divided into 8 CpG units (yellow highlight). (B) Quantitative CpG island methylation analysis: The colors of each circle represent the CpG island methylation level of each corresponding CpG unit. Yellow (~0% methylation), green (~50% methylation), and dark blue (~100% methylation). The white circles represent the missing data at a given CpG site. (C) Quantitative CpG island methylation analysis at CpG_3 and CpG_4 sites. The quantitative data represent the results of three independent experiments. (D), (E) and (F) Western blotting of the TFPI2, PCNA, MMP2, and MMP9 proteins in HASMCs treated with or without the 5-aza-dC methyltransferase inhibitor. 5-aza-dC seemed to recover the high glucose-stimulated TFPI2 depression in HASMCs. N = 3 in each group, *P < 0.05 vs. OC; #p < 0.05 vs. HG. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 2: Effects of TFPI2 on the Proliferation and Migration of High Glucose-stimulated HASMCs. (A) EDU staining of high-glucose stimulated HASMCs with TFPI2/pCDNA3.1 transfection (Scale bar: 100 μm). *P < 0.05 vs. OC + NT/pcDNA3.1; #p < 0.05 vs. HG + NT/pcDNA3.1. N = 3 per group. (B) Flow cytometry of the cell cycle. N = 3 in each group, *P < 0.05 vs. OC + NT/pcDNA3.1; #p < 0.05 vs. HG + NT/pcDNA3.1. (C) Transwell migration assays of high-glucose-stimulated HASMCs with TFPI2/pCDNA3.1 transfection. N = 3 in each group,*P < 0.05 vs. OC + NT/pcDNA3.1; #p < 0.05 vs. HG + NT/pcDNA3.1. (D) Western blotting of MMP2 and MMP9 protein expression. (E) Gelatin zymography of MMP2 and MMP9 activities. N = 3 in each group,*P < 0.05 vs. OC + NT/pcDNA3.1; #p < 0.05 vs. HG + NT/pcDNA3.1.
Fig 3: High levels of TFPI2 in the highly invasive melanoma cell line. (A) A Venn diagram shows overlaps of DEGs from three datasets. TFPI2 was consistently upregulated in the C918 cell line and metastasis (+) UM. (B) Western blotting shows higher levels of TFPI2 in C918 cells than in OCM-1A cells in whole cell lysate. (C) Representative IHC images stained for TFPI2 showing strikingly strong and weak expression of TFPI2 in xenografts of C918 and OCM-1A cells, respectively (upper panel). TFPI2-positive melanoma cells predominately disposed along the vascular channels in the coxenograft (C918 + OCM-1A) (lower panel). Scale bar 50 μm.
Fig 4: Prognostic significance of TFPI2 in melanoma patients. (A) Dot plot represents mRNA levels of TFPI2 in metastasis (+) vs. mestastasis (-) groups of TCGA-UM and TCGA-CM data, respectively. (B–D) Kaplan–Meier estimates of survival time for TCGA-UM, TCGA-CM, and TMU-CM cohort patients who were stratified into two groups: high and low levels of TFPI2.
Fig 5: Model of PARP1 Dependent Regulation Of TFPI2 Activity Enhancing Hyperglycemic-induced Neointimal Hyperplasia. DNA damage caused by hyperglycemia leads to PARP1 activity augmentation in VSMCs. Loss of PARP1 results in elevated TFPI2 gene expression through DNA hypomethylation. Activation of TFPI2 leads to decreased VSMCs proliferation and migration and suppressed excessive intimal hyperplasia in diabetes.
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