Journal: Arthritis and rheumatism

Article Title: Down-regulation of collagen and connective tissue growth factor expression with hepatocyte growth factor in lung fibroblasts from white scleroderma patients via two signaling pathways.

doi: 10.1002/art.22874

Figure Lengend Snippet: Figure 2. Effect of HGF on p44/p42 MAPK (ERK-1/2) phosphorylation in lung fibroblasts from systemic sclerosis (SSc) patients. A, HGF-induced ERK-1/2 phosphorylation was determined in cells treated for various time periods with HGF (50 ng/ml). Cell extracts were immunoblotted with anti–phospho–ERK-1/2 (ph-ERK-1/2) or anti–ERK-1/2 (as a control) (see Materials and Methods). B, Dose-dependence of ERK-1/2 phosphorylation was determined by adding HGF at varying doses (5–500 ng/ml) to SSc lung fibroblasts for 10 minutes. C, Lung fibroblasts were transfected with or without Grb2 siRNA, MMP-1 siRNA, and control siRNA or pretreated for 40 minutes with or without MEK-1/2 inhibitor U0126 (1 M), MMP inhibitor GM1489 (10 nM), or NF-B inhibitor BAY 11-7082 (10 M), then incubated with HGF (50 ng/ml) for 10 minutes. Anti-Grb2 was used to validate the knockdown effect of Grb2 siRNA. Cell culture supernatant was collected and subjected to enzyme-linked immunosorbent assay to measure the depletion of MMP-1 siRNA. Findings with control siRNA treatment were identical to findings with serum-free medium (lane 1). Quantitative results of densitometric analysis of immunoblots are also shown; values are the mean and SD from 3 independent experiments. P 0.05 versus unstimulated cells. See Figure 1 for other definitions.

Article Snippet: Recombinant human HGF and the Quantikine human proMMP-1 immunoassay kit were purchased from R&D Systems (Minneapolis, MN); an EnzoLytePlus 520 MMP-1 assay kit was obtained from AnaSpec (San Jose, CA); a NoShift transcription factor assay kit, NoShift NF- B (p65) reagents, and a NucBuster protein extraction kit were purchased from Novagen (Madison, WI); MEK-1/2 inhibitor U0126, MMP inhibitor GM1489, and NF- B inhibitor BAY 11-7082 were purchased from EMD Biosciences (San Diego, CA); Grb2 siRNA, MMP-1 siRNA, control siRNA-A, anti- CTGF, and fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti–type I collagen was obtained from Southern Biotechnology (Birmingham, AL); and anti– p44/p42 MAPK (anti–ERK-1/2), anti–phospho–p44/p42 MAPK, and anti-I B were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Control, Transfection, Incubation, Knockdown, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot