human il 27 hil 27  (R&D Systems)

Journal:

Article Title: IL-27 inhibits human osteoclastogenesis by abrogating RANKL-mediated induction of NFATc1 and suppressing proximal RANK signaling

doi: 10.1002/art.27200

Figure Lengend Snippet: Freshly isolated human CD14+ cells were cultured with MCSF (20ng/ml) for 48h and RANKL (40ng/ml) was added on Day 3 as described in Materials and Methods. IL-27 (3, 10, 30 and 100ng/ml) was added at the initiation of cultures (Day1) or later (Day 3 or Day 5). (A and B), TRAP positive multinucleated (> 3 nuclei) cells were counted 5 days after RANKL addition and representative data of one out of three independent experiments are shown as mean ±SD from triplicate wells of 96 well plates (* = p>0.05, ** = p<0.05 and *** = p<0.01. p values were calculated by Student's t test). C, Cathepsin K mRNA was measured by using real-time PCR and normalized relative to GAPDH expression. The means ±SD of triplicate determinants in a representative experiment of three independent experiments are shown; small SDs are not readily apparent because of large inductions. D (left panels), freshly isolated human monocytes were cultured as in A and osteoclast function was measured by a resorption pit formation assay. D (right panel), Human CD14+ monocytes were cultured with MCSF (20ng/ml) in the presence or absence of IL-27 (100ng/ml) for 2days. Cell viability was measured by MTT assay. Representative results of at least three independent experiments are shown.

Article Snippet: Recombinant hIL-27 (3-100ng/ml) (R&D Systems) was added either from the beginning of the culture and before RANKL (IL-27 pretreatment), simultaneously with RANKL on culture day 3, or after RANKL (on culture day 5).

Techniques: Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Tube Formation Assay, MTT Assay

Journal:

Article Title: IL-27 inhibits human osteoclastogenesis by abrogating RANKL-mediated induction of NFATc1 and suppressing proximal RANK signaling

doi: 10.1002/art.27200

Figure Lengend Snippet: A and B, Freshly isolated human CD14+ cells were cultured with MCSF (20ng/ml) in the presence or absence of IL-27 (100ng/ml) for 48h and then were stimulated with RANKL. C and D, Freshly isolated human CD14+ cells were cultured with MCSF (20ng/ml) for 48h and then were stimulated with RANKL (40ng/ml) in the presence or absence of IL-27 (100ng/ml). NFATc1 protein expression 24, 48 and 72h following RANKL stimulation was measured by immunoblotting (A and C). NFATc1 mRNA was measured using real-time PCR and normalized relative to GAPDH expression (B and D). Representative results of at least three independent experiments are shown.

Article Snippet: Recombinant hIL-27 (3-100ng/ml) (R&D Systems) was added either from the beginning of the culture and before RANKL (IL-27 pretreatment), simultaneously with RANKL on culture day 3, or after RANKL (on culture day 5).

Techniques: Isolation, Cell Culture, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal:

Article Title: IL-27 inhibits human osteoclastogenesis by abrogating RANKL-mediated induction of NFATc1 and suppressing proximal RANK signaling

doi: 10.1002/art.27200

Figure Lengend Snippet: Freshly isolated human CD14+ cells were cultured with MCSF (20ng/ml) in the presence or absence of IL-27 (100ng/ml) for 48h. Control and IL27-treated cells were stimulated with RANKL (40ng/ml) for 5, 10 and 15 minutes (A and B) or for 0.5, 1 and 3h (C). Immunoblotting was used to measure threonine 202/tyrosine 204 phosphorylation of Erk1/Erk2 and threonine 180/tyrosine 182 phoshorylation of p38 (A), serine 32 phosphorylation of IκBα and total IκBα (B), total TRAF6 (B), and nuclear c-Jun and TBP proteins (C). Representative results of at least five independent experiments are shown. D, RANK mRNA was measured in control and IL-27-treated cells using real-time PCR and normalized relative to GAPDH expression. The means ±SD of triplicate determinants in a representative experiment of three independent experiments are shown; small SDs are not readily apparent because of large inductions.

Article Snippet: Recombinant hIL-27 (3-100ng/ml) (R&D Systems) was added either from the beginning of the culture and before RANKL (IL-27 pretreatment), simultaneously with RANKL on culture day 3, or after RANKL (on culture day 5).

Techniques: Isolation, Cell Culture, Control, Western Blot, Phospho-proteomics, Real-time Polymerase Chain Reaction, Expressing

Journal:

Article Title: IL-27 inhibits human osteoclastogenesis by abrogating RANKL-mediated induction of NFATc1 and suppressing proximal RANK signaling

doi: 10.1002/art.27200

Figure Lengend Snippet: Freshly isolated human CD14+ cells were cultured with MCSF (20ng/ml) in the presence or absence of IL-27 (100ng/ml) for 24 and 48h. TREM-2 mRNA (A), OSCAR mRNA, SIRPβ1 mRNA and ILT7 mRNA (B) and DAP12 and FcRγ mRNA (C) was measured using real-time PCR and normalized relative to GAPDH expression. Representative results of at least four independent experiments are shown.

Article Snippet: Recombinant hIL-27 (3-100ng/ml) (R&D Systems) was added either from the beginning of the culture and before RANKL (IL-27 pretreatment), simultaneously with RANKL on culture day 3, or after RANKL (on culture day 5).

Techniques: Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Expressing

Journal:

Article Title: IL-27 inhibits human osteoclastogenesis by abrogating RANKL-mediated induction of NFATc1 and suppressing proximal RANK signaling

doi: 10.1002/art.27200

Figure Lengend Snippet: Freshly isolated CD14+ cells derived from synovial fluid of five RA patients were stimulated for 15min (A) or 3h (B) with hIFN-γ (100U/ml) or hIL-27 (100ng/ml). A, tyrosine 701 phosphorylation of STAT1 was measured by immunoblotting. Representative results of one out of five independent experiments are shown. B, CXCL10 mRNA expression was measured using real-time PCR and normalized relative to GAPDH expression (p<0.001 by paired Student's t test, n=5).

Article Snippet: Recombinant hIL-27 (3-100ng/ml) (R&D Systems) was added either from the beginning of the culture and before RANKL (IL-27 pretreatment), simultaneously with RANKL on culture day 3, or after RANKL (on culture day 5).

Techniques: Isolation, Derivative Assay, Phospho-proteomics, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal:

Article Title: IL-27 inhibits human osteoclastogenesis by abrogating RANKL-mediated induction of NFATc1 and suppressing proximal RANK signaling

doi: 10.1002/art.27200

Figure Lengend Snippet: A, Murine monocytes were stimulated for 5, 10 and 20min with mIFN-γ (100U/ml) or mIL-27 (100ng/ml). Immunoblotting was used to measure tyrosine 701 phosphorylation of STAT1 and tyrosine 705 phosphorylation of STAT3. (B-C) Bone marrow-derived osteoclast precursors obtained from C57BL/6J mice were cultured in the presence of MCSF (20ng/ml) and RANKL (80ng/ml) was added as described in Materials and Methods. IL-27 (1, 10 and 100ng/ml) was added 1 day before RANKL (Pretreatment) or simultaneously with RANKL. (B and C), TRAP positive multinucleated (> 3 nuclei) cells were counted 5 days after RANKL addition and representative data of one out of three independent experiments are shown as mean ±SD from triplicate wells of 96 well plates (* = p>0.05 and ** = p<0.05. p values were calculated by Student's t test). D, The expression of WSX-1 m-RNA was measured by PCR in freshly isolated human CD14+ and CD14- cells and in murine BMDM and splenocytes.

Article Snippet: Recombinant hIL-27 (3-100ng/ml) (R&D Systems) was added either from the beginning of the culture and before RANKL (IL-27 pretreatment), simultaneously with RANKL on culture day 3, or after RANKL (on culture day 5).

Techniques: Western Blot, Phospho-proteomics, Derivative Assay, Cell Culture, Expressing, Isolation