Journal:
Article Title: The Relative Timing of Exposure to Phagocytosable Particulates and to Osteoclastogenic Cytokines Is Critically Important in the Determination of Myeloid Cell Fate
doi: 10.4049/jimmunol.0902808
Figure Lengend Snippet: Phagocytosis inhibits expression of c-Fms. A, Preosteoclasts were cultured for 1 d with and without PMMA (20 particles/cell) or 100 ng/ml LPS in medium supplemented with 25 ng/ml M-CSF. RNA was extracted and c-Fms expression measured by qPCR. c-Fms mRNA levels expressed relative to GAPDH were normalized to the control sample without particles or LPS. n = 4. *p < 0.01 compared with control. B, Dose dependency of particle-mediated repression of c-Fms expression in preosteoclasts 1 d after particle addition. n = 3. *p < 0.01 compared with equivalent sample without particles. C, Preosteoclasts were treated with PMMA or LPS for 1, 3, or 24 h, and whole-cell protein lysates were analyzed by immunoblotting with anti–c-Fms Ab. Membranes were then reblotted with anti–β-actin Abs, and c-Fms levels were quantitated relative to β-actin. D, Preosteoclasts were preincubated with the MAPK inhibitors SB203580 (SB), U0216 (U0), and SP600125 (SP) for 30 min prior to addition of PMMA or silica (20 particles/cell). After 3 h, protein extracts were prepared and analyzed by immunoblotting as above. E, Preosteoclasts were pretreated with TAPI for 30 min prior to addition of LPS or particles. After 1 or 3 h, protein extracts were prepared and analyzed by immunoblotting. F, Preosteoclasts were pretreated with TAPI for 30 min and then treated with or without PMMA or LPS for 3 h, and c-Fms ectodomain shed into conditioned media was analyzed by ELISA. n = 3. *p < 0.01 compared with control; **p < 0.01 compared with equivalent samples without TAPI.
Article Snippet: The inhibitors used were SB203580 (p38, 10 μM), U0126 (MEK, 20 μM), SP600125 (JNK, 20 μM), and InSolution TNF-α protease inhibitor (TAPI) (TACE, 50 μM) from Calbiochem (San Diego, CA).
Techniques: Expressing, Cell Culture, Control, Western Blot, Enzyme-linked Immunosorbent Assay
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