Journal: Arthritis and rheumatism

Article Title: Impairment of the antifibrotic effect of hepatocyte growth factor in lung fibroblasts from African Americans: possible role in systemic sclerosis.

doi: 10.1002/art.22713

Figure Lengend Snippet: Figure 2. Levels of cytokines in bronchoalveolar lavage (BAL) fluid from Caucasian and African American patients with systemic sclerosis (SSc) and normal controls. Cytokine levels were significantly elevated in BAL fluid from African American control subjects as compared with Caucasian control subjects. A, Levels of hepatocyte growth factor (HGF), insulin-like growth factor binding protein 3 (IGFBP-3), osteoprotegerin (OPG), stem cell factor (SCF), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF) were measured by cytokine array analysis. Array images were scanned and analyzed with National Institutes of Health Image software. Positive controls (6 per membrane) were used to normalize the results from the different membranes being compared. Values are the mean and SEM of 24 separate arrays of BAL fluid samples from controls and 40 separate arrays from SSc patients. Variation between 2 identical cytokine spots ranged from 0% to 10% in duplicate experiments. B, Effect of selected cytokines on type I collagen expression in lung fibroblasts from Caucasian and African American normal subjects and SSc patients. Confluent cultures of lung fibroblasts were serum-starved for 24 hours and then incubated for 72 hours with recombinant transforming growth factor (TGF; 5 ng/ml), which was used as a positive control in studies of normal lung fibroblasts, or with HGF, IGFBP-3, OPG, SCF, TPO, or VEGF (50 ng/ml each). Serum-free medium (SFM) was used as a basal level for type I collagen. Cells were collected with lysis buffer and analyzed by Western blotting using anti–type I collagen antibody. Anti–-actin antibody was used as the sample loading control. Shown are representative immunoblots from 3 independent experiments.

Article Snippet: Confluent cultures of lung fibroblasts were serum-starved for 24 hours, followed by incubation for 72 hours with recombinant transforming growth factor (TGF ; 5 ng/ml) and/or HGF (50 ng/ml or concentrations indicated below), insulin-like growth factor binding protein 3 (IGFBP-3), osteoprotegerin (OPG), stem cell factor (SCF), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF), with or without neutralizing anti-human HGF receptor (c-Met) antibody (all purchased from R&D Systems).

Techniques: Control, Binding Assay, Software, Membrane, Expressing, Incubation, Recombinant, Positive Control, Lysis, Western Blot

Journal: Arthritis and rheumatism

Article Title: Impairment of the antifibrotic effect of hepatocyte growth factor in lung fibroblasts from African Americans: possible role in systemic sclerosis.

doi: 10.1002/art.22713

Figure Lengend Snippet: Figure 5. Modulation of the effects of hepatocyte growth factor (HGF) on connective tissue growth factor (CTGF) expression and type I collagen accumulation by anti–c-Met antibody treatment of lung fibroblasts from Caucasian patients with systemic sclerosis (SSc) and normal controls. Anti–c-Met antibody (Ab) diminished the effects of HGF on CTGF expression and collagen accumulation. Serum-starved lung fibroblasts were incubated for 72 hours with medium containing anti–c-Met antibody (2 g/ml), with or without HGF (50 ng/ml). Normal lung fibroblasts were left untreated or were additionally treated with transforming growth factor (TGF; 5 ng/ml). Anti–- actin antibody was used as the sample loading control. Shown are representative immunoblots from 3 independent experiments.

Article Snippet: Confluent cultures of lung fibroblasts were serum-starved for 24 hours, followed by incubation for 72 hours with recombinant transforming growth factor (TGF ; 5 ng/ml) and/or HGF (50 ng/ml or concentrations indicated below), insulin-like growth factor binding protein 3 (IGFBP-3), osteoprotegerin (OPG), stem cell factor (SCF), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF), with or without neutralizing anti-human HGF receptor (c-Met) antibody (all purchased from R&D Systems).

Techniques: Expressing, Incubation, Control, Western Blot

Journal: Arthritis and rheumatism

Article Title: Impairment of the antifibrotic effect of hepatocyte growth factor in lung fibroblasts from African Americans: possible role in systemic sclerosis.

doi: 10.1002/art.22713

Figure Lengend Snippet: Figure 6. Hepatocyte growth factor (HGF)–induced c-Met receptor phosphorylation in lung fibroblasts from Caucasian and African American patients with systemic sclerosis (SSc). Lung fibroblasts were cultured to confluence on 6-well plates and collected with lysis buffer. Twenty micrograms of protein per sample was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (4–20% gradient gels) and analyzed by Western blotting using anti–phospho–c-Met (Ph-C-Met) or anti–c-Met polyclonal antibodies (see Patients and Methods for details). Anti–-actin antibody was used as the sample loading control. Shown are representative immunoblots from 3 independent experiments. A, Levels of phosphor- ylated and total c-Met receptor in lung fibroblasts from Caucasian and African American SSc patients. B, Concentration-dependent phosphorylation of c-Met receptor in lung fibroblasts from Caucasian and African American SSc patients treated with HGF for 5 minutes at the indicated concentrations. C, Time course of c-Met receptor phosphorylation in lung fibroblasts from Caucasian and African American SSc patients after treatment with HGF (50 ng/ml) for various periods (left). Western blots from the time course experiments were analyzed by scanning densitometry using National Institutes of Health Image software (right). Values are the mean of 4 samples at each time point.

Article Snippet: Confluent cultures of lung fibroblasts were serum-starved for 24 hours, followed by incubation for 72 hours with recombinant transforming growth factor (TGF ; 5 ng/ml) and/or HGF (50 ng/ml or concentrations indicated below), insulin-like growth factor binding protein 3 (IGFBP-3), osteoprotegerin (OPG), stem cell factor (SCF), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF), with or without neutralizing anti-human HGF receptor (c-Met) antibody (all purchased from R&D Systems).

Techniques: Phospho-proteomics, Cell Culture, Lysis, Polyacrylamide Gel Electrophoresis, Western Blot, Control, Concentration Assay, Software

Journal: eLife

Article Title: Single nuclei RNA-seq of mouse placental labyrinth development

doi: 10.7554/eLife.60266

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Met Polyclonal antibody , R and D Systems , AF276 , IF (1:100).

Techniques: Isolation, Software