Journal: The Journal of pathology

Article Title: A 3D tri-culture system reveals that activin receptor-like kinase 5 and connective tissue growth factor drive human glomerulosclerosis

doi: 10.1002/path.4960

Figure Lengend Snippet: (A) MC 3D monoculture without the addition of TGFβ, and (B) in the presence of TGFβ, shown by H&E staining of paraffin-embedded sections. Human collagen type I/III (immunostained with an antibody that recognises both collagen type I and III) (C) and human collagen type IV (D) can be demonstrated in nodules by immunoperoxidase staining. (E) High-power view of MC nodule by scanning electron microscopy in cross section. (F) MC nodule count and collagen type I alpha1 (COL1a1), and collagen type IV alpha1 (COL4a1) RNA quantification (n=3). (G) Effect of ALK5 inhibition (Alk5i) on MC nodule formation (n=3). (H) Effect of SMAD2 or SMAD3 siRNA knockdown in MCs on nodule formation (n=3) with immunoblots demonstrating degree of knockdown. [DharmaFECT (DH), non-targeting siRNA control (siCP), siSMAD2 (siS2), siSMAD3 (siS3), total SMAD2 (tSMAD2), total SMAD3 (tSMAD3)]. Error bars represent 1 s.d. NS, not significant; ***, p<0.001; **, p<0.01: 2-tailed Student’s t test.

Article Snippet: After culture, matrices were embedded in HistoGel, fixed in 4% paraformaldehyde and paraffin wax-embedded for sectioning and staining for human collagen type IV (mouse anti-human collagen IV monoclonal antibody 2150–0121, Bio-Rad, CA, USA) and human collagen type I/III (rabbit anti-human collagen I/III polyclonal antibody 2150–2210, Bio-Rad, CA, USA).

Techniques: Staining, Immunoperoxidase Staining, Electron Microscopy, Inhibition, Western Blot

Journal:

Article Title: Iloprost suppresses connective tissue growth factor production in fibroblasts and in the skin of scleroderma patients

doi:

Figure Lengend Snippet: Normal (gray bars) and scleroderma (black bars) fibroblasts were grown to confluence on six-well plates. The experimental conditions were as follows: C, control with medium only; I, treated with 1 ng/ml Iloprost; T, treated with 10 ng/ml TGF-β; T+I, treated with 10 ng/ml TGF-β plus 1 ng/ml Iloprost added simultaneously. Media were removed after 24 hours for assay of (a) CTGF by ELISA, and (b) N-terminal propeptide of type I procollagen by ELISA. Values shown represent the means of six replicates of each experiment using cells derived from six patients with recent-onset diffuse scleroderma and from six healthy controls. *P < 0.05; **P < 0.001. (c) Normal human fibroblasts were grown to confluence and treated with 10 ng/ml TGF-β, with or without 1 ng/ml Iloprost and then lysed after 24 hours. CTGF was assayed by Western blot analysis. (d) In other experiments normal human fibroblasts were grown to confluence and treated with 10 ng/ml TGF-β with or without 1 ng/ml Iloprost. After 8 hours, the cells were lysed, and RNA was extracted for Northern blot analysis.

Article Snippet: The nitrocellulose filters were stained with primary Ab for 1 hour using the following primary Ab’s: rabbit polyclonal anti-human CTGF as above, rabbit anti-human collagen type I (759103R; BIODESIGN International, Kennebunk, Maine, USA), mouse monoclonal IgG anti–COX-1 (SC 7299; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) goat polyclonal IgG anti–COX-2 (SC-1746; Santa Cruz Biotechnology Inc.).

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot, Northern Blot

Journal:

Article Title: Iloprost suppresses connective tissue growth factor production in fibroblasts and in the skin of scleroderma patients

doi:

Figure Lengend Snippet: Normal human fibroblasts were grown to confluence in six-well plates and treated at baseline with TGF-β (gray bars) (10 ng/ml). After 12 hours, Iloprost (1 ng/ml) was added to the media of some of the cells (black bars). (a) Media were removed at various time points for type I collagen assay using Western blot analysis. (b) Additional assays were performed on the media to measure CTGF by ELISA.

Article Snippet: The nitrocellulose filters were stained with primary Ab for 1 hour using the following primary Ab’s: rabbit polyclonal anti-human CTGF as above, rabbit anti-human collagen type I (759103R; BIODESIGN International, Kennebunk, Maine, USA), mouse monoclonal IgG anti–COX-1 (SC 7299; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) goat polyclonal IgG anti–COX-2 (SC-1746; Santa Cruz Biotechnology Inc.).

Techniques: Collagen Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Iloprost suppresses connective tissue growth factor production in fibroblasts and in the skin of scleroderma patients

doi:

Figure Lengend Snippet: Normal human fibroblasts were grown to 50% confluence and infected with an adenovirus-encoding the cDNA for CTGF at 500 viral particles per cell. After 2 hours, the media were washed off and replaced with DMEM. (a) Subsequently, media were removed for assay of CTGF (gray bars) and type I collagen(black bars), by Western blot analysis. (b) The recombinant adenovirus encoding cDNA for human CTGF. LITR, left inverted terminal repeat; RITR, right inverted terminal repeat.

Article Snippet: The nitrocellulose filters were stained with primary Ab for 1 hour using the following primary Ab’s: rabbit polyclonal anti-human CTGF as above, rabbit anti-human collagen type I (759103R; BIODESIGN International, Kennebunk, Maine, USA), mouse monoclonal IgG anti–COX-1 (SC 7299; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) goat polyclonal IgG anti–COX-2 (SC-1746; Santa Cruz Biotechnology Inc.).

Techniques: Infection, Western Blot, Recombinant