Journal: Arthritis and rheumatism

Article Title: Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.

doi: 10.1002/art.23937

Figure Lengend Snippet: Figure 1. Effects of epigallocatechin-3-gallate (EGCG) on oncostatin M (OSM)–stimulated CCL2 production and MEK/ERK phosphorylation. A, Human bone marrow–derived osteoblasts were incubated for various periods of time with 10 ng/ml OSM, and the CCL2 mRNA levels were assayed by Northern blotting. B and C, Human bone marrow–derived osteoblasts (B) and MG-63 cells (C) were incubated for 8 hours with OSM, alone or in combination with 10 g/ml EGCG (3 hours before the addition of OSM), and CCL2 mRNA levels were subjected to Northern blotting. D and E, Cells were incubated for 24, 48, or 72 hours with OSM, alone or in combination with 10 g/ml EGCG (3 hours before the addition of OSM). CCL2 released into the culture media of human bone marrow–derived osteoblasts (D) and MG-63 (E) was quantified by enzyme-linked immunosorbent assay. Open bars indicate OSM; solid bars indicate OSM combined with EGCG. Values are the mean and SD results from 3 independent experiments. P 0.05 versus OSM at 0 hour; P 0.05. F, MG-63 cells were incubated for 20 minutes with OSM, alone or in combination with EGCG, and were assessed for phosphorylation at MEK-1/2 (Ser217/221) and ERK-1/2 (Thr202/Tyr204).

Article Snippet: Anti-human CCL2 antibody was obtained from R&D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Derivative Assay, Incubation, Northern Blot, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.

doi: 10.1002/art.23937

Figure Lengend Snippet: Figure 4. Effects of EGCG on OSM-induced activator protein 1 (AP-1)–DNA binding and AP-1–CCL2 promoter interaction in MG-63 cells. A–C, Cells were incubated for 30 minutes with 10 ng/ml OSM, alone or in combination with 10 g/ml EGCG (3 hours before the addition of OSM). The expression of c-Fos (A) and c-Jun (B) mRNA was determined by Northern blot analysis, and the respective protein levels were examined by Western blotting (C). D, Nuclear extracts from cells were subjected to electrophoretic mobility shift assay with supershift, using anti–c-Fos antibody. E, Cells were also subjected to chromatin immunoprecipitation (IP) assay with the indicated antibodies or with normal rabbit serum as control (mock). Immunoprecipitates from each sample, including input and mock, were analyzed by polymerase chain reaction using primers specific for the AP-1 binding region of human CCL2 promoter. Ac-H3 acetylated histone 3 (see Figure 1 for other definitions).

Article Snippet: Anti-human CCL2 antibody was obtained from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Expressing, Northern Blot, Western Blot, Electrophoretic Mobility Shift Assay, Chromatin Immunoprecipitation, Control, Polymerase Chain Reaction

Journal: Arthritis and rheumatism

Article Title: Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.

doi: 10.1002/art.23937

Figure Lengend Snippet: Figure 6. Immunolocalization of CCL2 and CD68 in the ankle joints of rats with collagen-induced arthritis. A–D, Sections from the control group. A, Pannus (P) formation and obliteration of joint space (JS). B, High-power view of A, showing erosion of cartilage and bone by pannus. C, Marked expression of CCL2 in osteoblasts (arrowheads) overlying osteolytic areas and mononuclear round cells (arrows). D, Clearly visible CD68 in macrophages (arrowheads) and osteoclasts (arrows) adjacent to resorption lacunae. E–H, Sections from the group treated with epigallocatechin- 3-gallate. E, Preservation of joint space, cartilage, and bone. F, High-power view of E, showing minimal inflammatory cell infiltration and cartilage erosion. G, Diminished numbers of CCL2 osteoblasts (arrowheads). H, Decreased infiltration of CD68 macrophages (arrowheads) and osteoclasts (arrows). (Hematoxylin and eosin staining in A, B, E, and F; avidinbiotinperoxidase staining in C, D, G, and H. (Original magnification 25 in A and E; 200 in B, C, D, F, G, and H.)

Article Snippet: Anti-human CCL2 antibody was obtained from R&D Systems (Minneapolis, MN).

Techniques: Control, Expressing, Preserving, Staining