Journal: Kidney international

Article Title: Migration of smooth muscle cells from the arterial anastomosis of arteriovenous fistulas requires Notch activation to form neointima

doi: 10.1038/ki.2015.73

Figure Lengend Snippet: In mice with CKD, FSP-1 and activated Notch1 are expressed in AVFs. A. FSP-1 mRNA was detected (RT-PCR) was 3.5-fold greater in AVFs of CKD mice vs. control. B. Expression of FSP-1 was detected by immunohistochemistry (n = 4 per group). C. Density analysis of FSP-1 levels from panel B. D & E. Co-immunostaining of SMA-α with FSP-1 (D) or SMC terminal differentiation marker SMMHC (E) in AVFs. F & G. Vimentin was co-immunostained with SMA-α (F) and FSP-1 (G) in AVFs. H. AVFs were prepared from control and CKD mice, Notch receptor and their target genes were detected by real time RT-PCR (*, p < 0.05). I. In AVFs from CKD mice, SMCs expressing N1ICD co-stained positively for FSP-1. The expression of N1ICD (green) and FSP-1 (red) were determined by double immunostaining (See arrows). Scale: 50 μm; L, lumen.

Article Snippet: The DNA was immunoprecipitated with the anti-N1ICD antibody and ChIP analysis was performed according to the manufacture’s protocol (Upstate Biotechnology, Lake Placid, NY) with some modification.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Immunohistochemistry, Immunostaining, Marker, Quantitative RT-PCR, Staining, Double Immunostaining

Journal: Kidney international

Article Title: Migration of smooth muscle cells from the arterial anastomosis of arteriovenous fistulas requires Notch activation to form neointima

doi: 10.1038/ki.2015.73

Figure Lengend Snippet: Notch mediates DNA binding activity in FSP-1 promoter. A. ChIP analysis of TGF-β1-induced DNA binding activity to the FSP-1 promoter of SMCs was evaluated by immunoprecipitation with N1ICD antibodies followed by PCR to detect the DNA binding activity. Representative data from 3 repeated experiments are shown. B. TGF-β1-induced DNA binding activity was inhibited by Notch inhibitors. SMCs pretreated with DAPT or infected with soluble Jagged1 before SMCs were treated with TGF-β1 for 2 h; DNA binding activity was detected by ChIP assay. C. SMCs infected with AdJagged1 (full length) in the presence/absence of Notch inhibitors, DAPT or soluble Jagged1 were used in ChIP assays. D & E. Relative density analysis of results from Panel B & C, respectively. F. Electrophoretic mobility shift assay (EMSA) of Notch-dependent binding activity induced in the FSP-1 promoter by TGF-β1 treatment. SMCs were examined with or without 30 min of pretreatment with 10 μM DAPT followed by 2h of TGF-â1 treatment. Nuclear proteins were incubated with the Dye-680 labeled probe (P6). G. SMCs infected with the Jagged1 adenovirus or vector for 24 h were examined by EMSA in 3 repeated experiments. H. A super-shift analysis of Jagged1-induced DNA binding activity to the FSP-1 promoter: SMCs were treated with vector or AdJagged1. Nuclear proteins from SMCs were incubated with the indicated antibodies for 1 h before the EMSA analysis was undertaken.

Article Snippet: The DNA was immunoprecipitated with the anti-N1ICD antibody and ChIP analysis was performed according to the manufacture’s protocol (Upstate Biotechnology, Lake Placid, NY) with some modification.

Techniques: Binding Assay, Activity Assay, Immunoprecipitation, Infection, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Plasmid Preparation

Journal: Kidney international

Article Title: Migration of smooth muscle cells from the arterial anastomosis of arteriovenous fistulas requires Notch activation to form neointima

doi: 10.1038/ki.2015.73

Figure Lengend Snippet: In failed AVFs from ESRD patients, FSP-1 is expressed in neointima cells. A. Morphology and SMA-α-positive cells from failed AVFs of ESRD patients (n =5). H & E (left panel) and SMA-α immunostaining (right panel) are shown. B. Characterization of neointima cells found in AVFs from ESRD patients. Cross sections of AVFs were immunostained for the FSP-1, PCNA, and SMA-α. (Red, double headed arrows show the neointima thickness, and green arrows indicate the media of AVFs). C. Coimmunostaining of FSP-1 and SMA-α in AVFs revealed that FSP-1 positive cells also express SMA-α. D. Activated Notch (N1ICD, brown color) is mainly located in nuclei of neointima cells in the failed AVFs of ESRD patients. E. In AVFs from ESRD patients, SMCs expressing N1ICD co-stained positively for FSP-1. The expression of N1ICD (green) and FSP-1 (red) were determined by double immunostaining; the double-head arrow indicates the neointima (red) and the smooth muscle layer of the vein (green). The red arrow points to cells stained positively for N1ICD and FSP-1. (N, neointima; M, media; L, lumen). Scale: 50 μm. F. Staining for H & E (Left panel) and Masson Trichome (Right panel) of human PTFE grafts are shown. Representative pictures from two PTFE grafts are shown. G. Co-immunofluorescent of SMA-α and FSP-1 in PTFE graft revealed that FSP-1 positive cells also express SMA-α. H. In PTFE graft from ESRD patients, expression of N1ICD and FSP-1 was detected by immunofluorescent staining with FSP-1 (n = 7).

Article Snippet: The DNA was immunoprecipitated with the anti-N1ICD antibody and ChIP analysis was performed according to the manufacture’s protocol (Upstate Biotechnology, Lake Placid, NY) with some modification.

Techniques: Immunostaining, Expressing, Staining, Double Immunostaining